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• W2091044171 abstract "Mineral crystal nucleation in UMR 106-01 osteoblastic cultures occurs within 15–25-μm extracellular vesicle-containing biomineralization foci (BMF) structures. We show here that BAG-75 and BSP, biomarkers for these foci, are specifically enriched in laser capture microscope-isolated mineralized BMF as compared with the total cell layer. Unexpectedly, fragments of each protein (45–50 kDa in apparent size) were also enriched within captured BMF. When a series of inhibitors against different protease classes were screened, serine protease inhibitor 4-(2-aminoethyl)benzenesulfonylfluoride HCl (AEBSF) was the only one that completely blocked mineral nucleation within BMF in UMR cultures. AEBSF appeared to act on an osteoblast-derived protease at a late differentiation stage in this culture model just prior to mineral deposition. Similarly, mineralization of bone nodules in primary mouse calvarial osteoblastic cultures was completely blocked by AEBSF. Cleavage of BAG-75 and BSP was also inhibited at the minimum dosage of AEBSF sufficient to completely block mineralization of BMF. Two-dimensional SDS-PAGE comparisons of AEBSF-treated and untreated UMR cultures showed that fragmentation/activation of a limited number of other mineralization-related proteins was also blocked. Taken together, our results indicate for the first time that cleavage of BAG-75 and BSP by an AEBSF-sensitive, osteoblast-derived serine protease is associated with mineral crystal nucleation in BMF and suggest that such proteolytic events are a permissive step for mineralization to proceed. Mineral crystal nucleation in UMR 106-01 osteoblastic cultures occurs within 15–25-μm extracellular vesicle-containing biomineralization foci (BMF) structures. We show here that BAG-75 and BSP, biomarkers for these foci, are specifically enriched in laser capture microscope-isolated mineralized BMF as compared with the total cell layer. Unexpectedly, fragments of each protein (45–50 kDa in apparent size) were also enriched within captured BMF. When a series of inhibitors against different protease classes were screened, serine protease inhibitor 4-(2-aminoethyl)benzenesulfonylfluoride HCl (AEBSF) was the only one that completely blocked mineral nucleation within BMF in UMR cultures. AEBSF appeared to act on an osteoblast-derived protease at a late differentiation stage in this culture model just prior to mineral deposition. Similarly, mineralization of bone nodules in primary mouse calvarial osteoblastic cultures was completely blocked by AEBSF. Cleavage of BAG-75 and BSP was also inhibited at the minimum dosage of AEBSF sufficient to completely block mineralization of BMF. Two-dimensional SDS-PAGE comparisons of AEBSF-treated and untreated UMR cultures showed that fragmentation/activation of a limited number of other mineralization-related proteins was also blocked. Taken together, our results indicate for the first time that cleavage of BAG-75 and BSP by an AEBSF-sensitive, osteoblast-derived serine protease is associated with mineral crystal nucleation in BMF and suggest that such proteolytic events are a permissive step for mineralization to proceed. Bone is a vascularized tissue that uniquely becomes mineralized as part of its developmental program (1Olsen B.R. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. 2006; : 1-6Google Scholar). Mineralized bone serves essential vertebrate functions, including structural support, reversible storage for calcium and phosphorus, and as a reservoir for toxic metals and carbonate (2Dempster D.W. Lian J.B. Goldring S.R. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. 2006; : 7-11Google Scholar). Bone tissue is composed of osteoid; osteoblasts, which produce and mineralize new bone; osteoclasts, which resorb bone; and osteocytes, mature osteoblasts that maintain bone viability (1Olsen B.R. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. 2006; : 1-6Google Scholar, 2Dempster D.W. Lian J.B. Goldring S.R. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. 2006; : 7-11Google Scholar, 3Aubin J.E. Lian J.B. Stein G.S. Goldring S.R. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. 2006; : 20-29Google Scholar). Osteoid is a type I collagen-rich extracellular matrix enriched in acidic noncollagenous proteins (4Gehron Robey P. Boskey A.L. Lian J.B. Goldring S.R. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. 2006; : 12-19Google Scholar). Using fetal rat calvaria cell cultures, Bellows et al. (5Bellows C.G. Aubin J.E. Heersche J.N. Bone Miner. 1991; 14: 27-40Abstract Full Text PDF PubMed Scopus (334) Google Scholar) showed that osteoid is unmineralized when initially deposited, and mineral crystals form within nodular structures over the following 48–72 h. Bone matrices can be classified as lamellar, based on a highly organized layered structure, or woven bone. Woven bone is formed during embryonic development, fracture healing, and at sites receiving mechanical stimulation in excess of 3,000 microstrain (6Turner C.H. Akhter M.P. Raab D.M. Kimmel D.B. Recker R.R. Bone (Elmsford). 1991; 12: 73-79Crossref PubMed Scopus (205) Google Scholar); lamellar bone replaces woven bone later in development. The question of whether bone mineralization is under direct osteoblastic control or whether it is purely a passive chemical process is under active investigation. Schinke et al. (7Schinke T. McKee M.D. Karsenty G. Nat. Genet. 1999; 21: 150-151Crossref PubMed Scopus (126) Google Scholar) have proposed that calcification reactions in vivo are passive physiochemical processes occurring readily where local mineralization inhibitors are overwhelmed. In support of this hypothesis, Murshed et al. (8Murshed M. Harmey D. Millan J.L. McKee M.D. Karsenty G. Genes Dev. 2005; 19: 1093-1104Crossref PubMed Scopus (489) Google Scholar) produced a calcified dermal layer in transgenic mice expressing alkaline phosphatase in skin under the control of the type I collagen α chain promoter (2Dempster D.W. Lian J.B. Goldring S.R. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. 2006; : 7-11Google Scholar). Similarly, Luo et al. (9Luo G. Ducy P. McKee M.D. Pinero G.J. Loyer E. Behringer R.R. Karsenty G. Nature. 1997; 386: 78-81Crossref PubMed Scopus (1764) Google Scholar) and Murshed et al. (10Murshed M. Schinke T. McKee M.D. Karsenty G. J. Cell Biol. 2004; 165: 625-630Crossref PubMed Scopus (414) Google Scholar) showed that matrix GLA protein is a passive local inhibitor of vascular calcification because deficient mice calcify their thoracic aorta. The latter approach emphasizes the formation of hydroxyapatite crystals as the primary experimental outcome. A second view focuses on the active role of local extracellular nucleation complexes such as biomineralization foci (11Gorski J.P. Wang A. Lovitch D. Law D. Powell K. Midura R.J. J. Biol. Chem. 2004; 279: 25455-25463Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 12Midura R.J. Wang A. Lovitch D. Law D. Powell K. Gorski J.P. J. Biol. Chem. 2004; 279: 25464-25473Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), crystal ghosts (13Bonucci E. Calcif. Tissue Int. 1979; 29: 181-182Crossref PubMed Scopus (7) Google Scholar, 14Bonucci E. Silvestrini G. di Grezia R. Connect. Tissue Res. 1989; 22: 43-50Crossref PubMed Scopus (12) Google Scholar), matrix vesicles (15Anderson H.C. Clin. Orthop. Relat. Res. 1995; 314: 266-280PubMed Google Scholar), and the hole regions of collagen fibrils (16Lee D.D. Glimcher M.J. Connect. Tissue Res. 1989; 21: 247-257Crossref PubMed Scopus (15) Google Scholar) with matrix vesicles (17Arsenault A.L. J. Electron Microsc. Technol. 1991; 18: 262-268Crossref PubMed Scopus (16) Google Scholar, 18Wiesmann H.P. Meyer U. Plate U. Hohling H.J. Int. Rev. Cytol. 2005; 242: 121-156Crossref PubMed Scopus (97) Google Scholar) or with extracellular matrix phosphoproteins (12Midura R.J. Wang A. Lovitch D. Law D. Powell K. Gorski J.P. J. Biol. Chem. 2004; 279: 25464-25473Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 19Gericke A. Qin C. Spevak L. Fujimoto Y. Butler W.T. Sorensen E.S. Boskey A.L. Calcif. Tissue Int. 2005; 77: 45-54Crossref PubMed Scopus (252) Google Scholar, 20Hunter G.K. Goldberg H.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8562-8565Crossref PubMed Scopus (580) Google Scholar). We have proposed that mineralization can be divided into a cell-mediated nucleation phase within BMF, 2The abbreviations used are: BMFbiomineralization fociAEBSF4-(2-aminoethyl)benzenesulfonylfluoride HClFBSfetal bovine serumBSPbone sialoproteinBAG-75bone acidic glycoprotein-75BGPβ-glycerol phosphateBSAbovine serum albumin+CLtotal cell layer extract from BGP-treated cultures–CLtotal cell layer extract from cultures not treated with BGPMAAMaackia amurensis agglutininMS/MStandem mass spectrometryLCliquid chromatographySKI-1subtilisin kexin isozyme-1LCMlaser capture microscope1,25D3-MARRS1,25-vitamin D3-membrane-associated rapid-response steroid-binding proteinCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidCAPS3-(cyclohexylamino)propanesulfonic acidMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideMEMminimal essential mediumMAAMaackia amurensis agglutinin. followed by passive growth and expansion of these initial crystals (11Gorski J.P. Wang A. Lovitch D. Law D. Powell K. Midura R.J. J. Biol. Chem. 2004; 279: 25455-25463Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 12Midura R.J. Wang A. Lovitch D. Law D. Powell K. Gorski J.P. J. Biol. Chem. 2004; 279: 25464-25473Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). In this model, once the initial crystals reach sufficient size and number, the BMF barrier function is abrogated, facilitating the passive growth and expansion of the initial mineral phase into the larger, territorial collagenous matrix. The latter research focuses on the in vivo functionality of the mineralized bone product (10Murshed M. Schinke T. McKee M.D. Karsenty G. J. Cell Biol. 2004; 165: 625-630Crossref PubMed Scopus (414) Google Scholar, 11Gorski J.P. Wang A. Lovitch D. Law D. Powell K. Midura R.J. J. Biol. Chem. 2004; 279: 25455-25463Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 12Midura R.J. Wang A. Lovitch D. Law D. Powell K. Gorski J.P. J. Biol. Chem. 2004; 279: 25464-25473Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 13Bonucci E. Calcif. Tissue Int. 1979; 29: 181-182Crossref PubMed Scopus (7) Google Scholar, 14Bonucci E. Silvestrini G. di Grezia R. Connect. Tissue Res. 1989; 22: 43-50Crossref PubMed Scopus (12) Google Scholar, 15Anderson H.C. Clin. Orthop. Relat. Res. 1995; 314: 266-280PubMed Google Scholar, 16Lee D.D. Glimcher M.J. Connect. Tissue Res. 1989; 21: 247-257Crossref PubMed Scopus (15) Google Scholar, 17Arsenault A.L. J. Electron Microsc. Technol. 1991; 18: 262-268Crossref PubMed Scopus (16) Google Scholar, 18Wiesmann H.P. Meyer U. Plate U. Hohling H.J. Int. Rev. Cytol. 2005; 242: 121-156Crossref PubMed Scopus (97) Google Scholar, 19Gericke A. Qin C. Spevak L. Fujimoto Y. Butler W.T. Sorensen E.S. Boskey A.L. Calcif. Tissue Int. 2005; 77: 45-54Crossref PubMed Scopus (252) Google Scholar). In this context, hydroxyapatite crystal formation is envisioned to occur in a manner that facilitates subsequent vascular access to the crystals and placement of crystals within the organic matrix so as to facilitate mechanical support for organs, joints, muscles, and tendons. biomineralization foci 4-(2-aminoethyl)benzenesulfonylfluoride HCl fetal bovine serum bone sialoprotein bone acidic glycoprotein-75 β-glycerol phosphate bovine serum albumin total cell layer extract from BGP-treated cultures total cell layer extract from cultures not treated with BGP Maackia amurensis agglutinin tandem mass spectrometry liquid chromatography subtilisin kexin isozyme-1 laser capture microscope 1,25-vitamin D3-membrane-associated rapid-response steroid-binding protein 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid 3-(cyclohexylamino)propanesulfonic acid 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide minimal essential medium Maackia amurensis agglutinin. Bone osteoid is enriched in phosphoproteins, acidic glycoproteins, and proteoglycans, some of which like BSP or its fragments are nucleators of hydroxyapatite crystals (20Hunter G.K. Goldberg H.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8562-8565Crossref PubMed Scopus (580) Google Scholar, 21Goldberg H.A. Warner K.J. Stillman M.J. Hunter G.K. Connect. Tissue Res. 1996; 35: 385-392Crossref PubMed Scopus (73) Google Scholar). We have shown that phosphoglycoprotein BAG-75 expression delineates future extracellular sites of mineralization in vivo within woven bone and in vitro termed BMF (11Gorski J.P. Wang A. Lovitch D. Law D. Powell K. Midura R.J. J. Biol. Chem. 2004; 279: 25455-25463Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 12Midura R.J. Wang A. Lovitch D. Law D. Powell K. Gorski J.P. J. Biol. Chem. 2004; 279: 25464-25473Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). BMF are 15–25-μm spherical extracellular structures containing several sizes of vesicles (15Anderson H.C. Clin. Orthop. Relat. Res. 1995; 314: 266-280PubMed Google Scholar), which are sites of the first mineral crystals in the UMR osteoblastic model (12Midura R.J. Wang A. Lovitch D. Law D. Powell K. Gorski J.P. J. Biol. Chem. 2004; 279: 25464-25473Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Following plating, UMR cells proliferate and differentiate over the first 60–64 h and attain a competency to initiate mineralization in BMF, if supplemented with a phosphate source (22Wang A. Martin J.A. Lembke L.A. Midura R.J. J. Biol. Chem. 2000; 275: 11082-11091Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). BSP has also been localized to mineralizing nodules termed crystal ghost aggregates in rat bone, which are analogous to BMF (13Bonucci E. Calcif. Tissue Int. 1979; 29: 181-182Crossref PubMed Scopus (7) Google Scholar, 14Bonucci E. Silvestrini G. di Grezia R. Connect. Tissue Res. 1989; 22: 43-50Crossref PubMed Scopus (12) Google Scholar, 23Bianco P. Riminucci M. Silvestrini G. Bonucci E. Termine J.D. Fisher L.W. Robey P.G. J. Histochem. Cytochem. 1993; 41: 193-203Crossref PubMed Scopus (123) Google Scholar). BSP is incorporated into BMF just prior the appearance of mineral crystals (12Midura R.J. Wang A. Lovitch D. Law D. Powell K. Gorski J.P. J. Biol. Chem. 2004; 279: 25464-25473Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 22Wang A. Martin J.A. Lembke L.A. Midura R.J. J. Biol. Chem. 2000; 275: 11082-11091Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Based on these findings, we proposed that BMF structures function in an active mineralization process initiated and controlled by osteoblastic cells. To better understand the role of BMF in bone mineral nucleation, we have begun to characterize the proteome of mineralized BMF isolated by laser capture microscopy. Our results show that isolated BMF are not only physically enriched in BAG-75 and BSP but also fragments of each. Screening inhibitors of the different classes of proteases revealed for the first time that serine protease inhibitor AEBSF completely blocked cleavage of BAG-75 and BSP, as well as mineral crystal nucleation within BMF. Two-dimensional SDS-PAGE comparisons of AEBSF-treated and control cultures suggested that activation of procollagen processing may also be inhibited. Taken together, our results demonstrate an association between serine protease cleavage of mineral nucleator BSP and mineral crystal nucleation within biomineralization foci and mineralization nodules. Antibodies were from several sources as follows: nonimmune rabbit IgG (EMD Biosciences), anti-BAG-75 (number 503) (anti-peptide antibody) rabbit serum (24Gorski J.P. Griffin D. Dudley G. Stanford C. Thomas R. Huang C. Lai E. Karr B. Solursh M. J. Biol. Chem. 1990; 265: 14956-14963Abstract Full Text PDF PubMed Google Scholar); anti-BAG-75 (number 504) (anti-protein antibody) rabbit serum (24Gorski J.P. Griffin D. Dudley G. Stanford C. Thomas R. Huang C. Lai E. Karr B. Solursh M. J. Biol. Chem. 1990; 265: 14956-14963Abstract Full Text PDF PubMed Google Scholar); anti-bone sialoprotein LF-100 antiserum (Larry Fisher, NIDCR, National Institutes of Health); and monoclonal anti-BSP (WV1D1(9C5)) antibody (NIH Developmental Studies Hybridoma Bank, University of Iowa). Cell Culture—UMR 106-01 BSP cells were passaged and cultured at 37 °C and 5% carbon dioxide as described previously (12Midura R.J. Wang A. Lovitch D. Law D. Powell K. Gorski J.P. J. Biol. Chem. 2004; 279: 25464-25473Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 22Wang A. Martin J.A. Lembke L.A. Midura R.J. J. Biol. Chem. 2000; 275: 11082-11091Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and updated briefly here. Cells were seeded at a density of 1.0 × 105 cells/cm2 in Growth Medium (Eagle's MEM supplemented with Earle's salts, 1% nonessential amino acids (Sigma), 10 mm HEPES (pH 7.2), and 10% fetal bovine serum (Hyclone)). After 24 h, the medium was exchanged with Growth Medium containing 0.5% BSA (catalog number A-1933, Sigma) instead of FBS. Sixty four hours after plating, the culture medium was exchanged with Mineralization Media (Growth Medium containing either 0.1% BSA or 10% fetal bovine serum and 7 mm BGP). Cultures were then incubated for an additional 24 h, at the end of which (88 h) the cells were either subjected to MTT assay or fixed in 70% ethanol and then extracted for protein. In some experiments, protease inhibitors, including serine protease inhibitor AEBSF [(4-(2-aminoethyl)-benzenesulfonylfluoride HCl)] (EMD Biosciences), were added to cultures at 64 h after plating in Mineralization Media. Alternatively, AEBSF was added at 44 h after plating; inhibitor was then removed and exchanged for Mineralization Media at 64 h and the amount of mineralization analyzed at 88 h. Primary mouse osteoblasts were isolated from calvaria of 5–7-day-old mice using a modification of the method described previously (25Kalajzic I. Kalajzic Z. Kaliterna M. Gronowicz G. Clark S.H. Lichtler A.C. Rowe D. J. Bone Miner. Res. 2002; 17: 15-25Crossref PubMed Scopus (323) Google Scholar, 26Kalajzic I. Staal A. Yang W.P. Wu Y. Johnson S.E. Feyen J.H. Krueger W. Maye P. Yu F. Zhao Y. Kuo L. Gupta R.R. Achenie L.E. Wang H.W. Shin D.G. Rowe D.W. J. Biol. Chem. 2005; 280: 24618-24626Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Briefly, the calvaria were aseptically harvested, and four sequential 20-min digests were performed in 0.05% trypsin, 0.2% collagenase in Hanks' balanced salt solution. Fractions 2–4 were pooled, centrifuged, and resuspended in α-MEM containing 10% fetal bovine serum, 2 mml-glutamine, 100 units/ml penicillin, and 30 μg/ml gentamicin (α-Growth Medium). 2 × 106 cells were plated per T-75-cm2 flask and allowed to reach confluency (3–4 days). Confluent flasks were then trypsinized and plated into 12- or 24-well culture dishes for experiments at a density of 20,000 cells per cm2 growth area using media and supplements as described above. At confluency, the media were changed to α-MEM containing 5% FBS, 50 μg/ml ascorbic acid, 5 mm BGP, and other supplements as described above. BGP was omitted from some wells that served as an un-mineralized control. To test the effect of AEBSF, identical duplicate cultures were treated on days 3, 6, or 9 with 0.003 to 0.1 mm AEBSF. Phase contrast images were taken of living cultures on days 3–12. On day 12 after plating, one set of cultures was incubated with MTT as described below to determine cell viability. A second set of cultures was fixed on day 12 with 70% ethanol and processed for quantitative Alizarin red S staining as described below. MTT Assay—Culture wells were washed with Eagle's MEM supplemented with Earle's salts and then incubated with a solution of 0.5 mg/ml MTT in Eagle's MEM for 1–2 h at 37 °C (27Mosmann T. J. Immunol. Methods. 1983; 65: 55-63Crossref PubMed Scopus (46884) Google Scholar). Residual MTT solution was removed; the cells were disrupted by mixing briefly with dimethyl sulfoxide, and free reduced dye was read at 490 or 540 nm in a spectrophotometer. Quantitation of Mineralization—After fixation in 70% ethanol, the cell layer was rinsed and stained with Alizarin red S dye as described previously (22Wang A. Martin J.A. Lembke L.A. Midura R.J. J. Biol. Chem. 2000; 275: 11082-11091Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The same procedure was also used for serum-depleted cultures with the following modified washing protocol, e.g. the stained cell layer was rinsed once with 1 mm HEPES in nanopure water. A standard curve for Alizarin red S dye was constructed for each analysis, and amount of bound dye/culture well determined. Statistical Methods—All statistical tests were performed using SigmaStat 3.1 software (Systat Software, Inc.). A one-way analysis of variance test was used to determine whether a statistical difference existed between the viability of UMR-106-01 cultures or the amount of mineral deposited. Subsequent pairwise multiple comparison tests were performed with the Student-Newman-Keuls or the Kruskal-Wallis method. Extraction of Cell Layer Fraction; One-step Method—Cells were dislodged by scraping and then extracted with 75 mm potassium phosphate buffer (pH 7.2), containing 10 mm CHAPS, 75 mm sodium chloride, 50 mm tetrasodium EDTA, 10 mm benzamidine hydrochloride, 2 mm dithiothreitol, and 0.02% sodium azide for 1 h at 4°C. Each extract was then homogenized briefly using a motorized pestle and clarified by ultracentrifugation at 30,000 rpm for 1 h at 4°C in an SW 50.1 rotor prior to use. Conditioned media were immediately heated at 95 °C for 5 min to inactivate protease activity and frozen at –80 °C until analyzed. Extraction of Cell Layer; Two-step Method—During the final 24-h mineralization period, cells were grown in BSA-free, serum-free media conditions to reduce the amount of BSA in fractions used for two-dimensional gel electrophoresis. Media were removed from each flask, heated at 95 °C for 5 min, dialyzed against 5% acetic acid, and lyophilized to dryness. Cell layers were first extracted without mixing for 2 h at 4 °C in 0.05 m Tris acetate buffer (pH 7.5) containing 0.15 m NaCl, 0.05 m EDTA, and 0.02% sodium azide; extracts were then inactivated at 95 °C for 5 min, dialyzed against 5% HAc, and lyophilized to dryness. The residual cell layer was next dislodged by scraping and extracted overnight at 4 °C by slow mixing with 0.1 m Tris acetate buffer (pH 7.5) containing 8 m urea, 2% (w/v) CHAPS, and 0.02% sodium azide. Urea extracts were homogenized and clarified by ultracentrifugation at 30,000 rpm for 1 h at 4 °C in an SW 50.1 rotor prior to use in two-dimensional gel electrophoresis. Western Blotting Chemiluminescence Detection—Cell layer extracts and media fractions prepared as described above were electrophoresed under reducing conditions on 4–20% linear gradient gels (ISC BioExpress) according to Laemmli (28Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and electroblotted onto polyvinylidene difluoride membranes (Millipore Corp.) for 2 h at 100 V. The transfer buffer was composed of 10 mm CAPS buffer (pH 11.0) containing 10% methanol. Blots were processed essentially as described previously (29Gorski J.P. Liu F.T. Artigues A. Castagna L.F. Osdoby P. J. Biol. Chem. 2002; 277: 18840-18848Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Immunoblotting with digoxygenin-MAA lectin followed a procedure described earlier (29Gorski J.P. Liu F.T. Artigues A. Castagna L.F. Osdoby P. J. Biol. Chem. 2002; 277: 18840-18848Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) except that a secondary horseradish peroxidase-conjugated anti-digoxygenin antibody was used for chemiluminescent detection. Films were digitized using a flat bed scanner. To detect directly acidic bone glycoproteins or phosphoproteins, some gels were stained with Stains All (29Gorski J.P. Liu F.T. Artigues A. Castagna L.F. Osdoby P. J. Biol. Chem. 2002; 277: 18840-18848Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Laser Capture Microscopy—UMR cells were grown as usual on Fisher Plus microscope slides (Fisher), fixed, and stained with Alizarin red S dye. Immediately prior to laser capture, slides were dehydrated through a graded series of ethanol washes and xylene rinses. Dried slides were stored at –20 °C in a sealed box with desiccant until used. Mineralized BMF were collected onto standard caps using an Arturus Pixel IIe microscope. Collection films were pooled and stored in 70% ethanol at –20 °C until ∼6200 BMF were collected. LCM-captured BMF were then mixed in 70% ethanol to dislodge the purple-stained particles that were then microcentrifuged to remove the ethanol. BMF pellets were extracted twice sequentially over a 2-day period at 4 °C with 1.1 ml of 0.1 m Tris acetate buffer (pH 7.8) containing with 0.5% octyl glucoside, 0.05% SDS, 0.05 m EDTA, and 0.02% sodium azide. Extracts were then dialyzed first against 0.01 m Tris acetate buffer (pH 7.8) containing 8 m urea, 0.05% SDS, 0.1% octyl glucoside, 0.05 m EDTA, and second against 0.01 m Tris acetate buffer (pH 7.8) containing 8 m urea, 0.05% SDS, and 0.1% octyl glucoside. Controls consisted of glass slides containing the total cell layer fractions from +BGP or –BGP cultures; control slides were extracted using a similar protocol. The resultant dialyzed extracts were used for comparative blotting studies where identical protein amounts were loaded per gel lane. Protein Determination—Protein concentration of BMF extracts was determined using the NonInterfering Protein Assay by Geno-Technology Inc. (St. Louis, MO). Mass Spectrometric Analyses—Protein bands and spots were detected by staining with Coomassie Blue G dye or with Sypro Ruby dye according to the manufacturer's instructions (Bio-Rad). Excised gel bands/spots were reduced and alkylated followed by digestion with trypsin for 6–16 h (30Keightley J.A. Shang L. Kinter M. Mol. Cell. Proteomics. 2004; 3: 167-175Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Peptides were extracted and subjected to reverse phase capillary liquid chromatography-mass spectrometry with a linear 2–70% acetonitrile gradient over 45 min in 50 mm acetic acid, in a 50 μm inner diameter × 7 mm Phenomenex C18 Jupiter Proteo capillary column. The column eluted directly into an LTQ linear ion trap mass spectrometer as described previously (30Keightley J.A. Shang L. Kinter M. Mol. Cell. Proteomics. 2004; 3: 167-175Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The instrument was operated in the data-dependent mode in which one mass spectrum and eight collision-induced dissociation spectra were acquired per cycle. The data were analyzed using Mascot protein identification software (Matrix Science), with manual inspection of data base matches for validation. The Mascot identification program (Matrix Science) uses a statistical method to assess the validity of a match (31Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6814) Google Scholar). Criteria used for protein identifications include matching the peptide based on the following: 1) the precursor (peptide) mass, and 2) MS/MS fragment masses present in the scan, coinciding with the predicted masses of peptides (and peptide fragment masses) from a data base entry. Protein searches are currently based on comparison to all or a subset of (rodent, for example) the sequences present in the MSDB data base, filename MSDB_20050227.fasta (February 27th, 2005 version). Protein identifications made contain at least two peptides match in the MS/MS scans that meets or exceeds the threshold values for a 95% confidence level. Two-dimensional PAGE—Gels were run according to the method of Witzmann et al. (32Witzmann F.A. Clack J.W. Geiss K. Hussain S. Juhl M.J. Rice C.M. Wang C. Electrophoresis. 2002; 23: 2223-2232Crossref PubMed Scopus (32) Google Scholar) and stained with either colloidal Coomassie Blue G, Pro-Q Emerald 300 glycoprotein stain (Invitrogen), or Pro-Q Diamond phosphoprotein stain (Invitrogen). PD-Quest (Bio-Rad) software was used to digitally analyze the colloidal Coomassie Blue G-stained gels comparing AEBSF-treated with nontreated cell layer and media fractions to identify proteins differentially expressed in one condition versus another. Mineralization of UMR Osteoblastic Cells Is Unchanged in Serum-depleted Conditions—To limit contamination by serum proteins in isolated BMF, we tested whether use of serum-free conditions would affect the amount or morphology of mineralization in UMR cultures. No differences were noted in the amount or morphology of mineralized BMF when conditions of serum depletion were compared with serum-replete conditions (compare Fig. 1, A versus C). As expected (22Wang A. Martin J.A. Lembke L.A. Midura R.J. J. Biol. Chem. 2000; 275: 11082-11091Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), few mineral crystals are evident when BGP is omitted (Fig. 1B). Quantitation of the amount of Alizarin red stain bound per well also revealed no significant differences (not shown). Manual counts of mineralized BMF formed under serum-containing and serum-depleted conditions showed no statistical difference (103 foci/cm2 ± 6.56 S.D. versus 105 mineralized foci/cm2 ± 6.08 S.D., p = 0.486 using one-way analysis of var" @default.
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• W2091044171 date "2007-09-01" @default.
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• W2091044171 title "Association of Specific Proteolytic Processing of Bone Sialoprotein and Bone Acidic Glycoprotein-75 with Mineralization within Biomineralization Foci" @default.
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• W2091044171 doi "https://doi.org/10.1074/jbc.m701332200" @default.
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