Bone acidic glycoprotein-75 is a major synthetic product of osteoblastic cells and localized as 75- and/or 50-kDa forms in mineralized phases of bone and growth plate and in serum

Anti-peptide and anti-protein antisera were produced which both recognize bone acidic glycoprotein-75 (Mr = 75,000) and an apparent fragment or biosynthetic intermediate (Mr = 50,000) in calcified tissues and/or serum. A fragment-precursor relationship is suggested from the fact that closely spaced doublet polypeptides of Mr = 50,000 could be produced by proteolysis of the purified protein upon long term storage. No reactivity was detected with osteopontin, bone sialoprotein, or small bone proteoglycans. Bone acidic glycoprotein-75 represents 0.5-1% of the total radiolabeled proteins synthesized by explant cultures of neonatal calvaria or growth plate, by calvarial outgrowth cultures, and by rat osteosarcoma cells. Amounts produced by explant cultures and calvarial outgrowth cultures were similar to that for osteopontin, a major product of osteoblasts. In osteosarcoma cultures, 80% of labeled antigens were associated with the cell layer fraction wherein specific immunoprecipitation pelleted Mr = 50,000 and 75,000 sized antigens. Bone acidic glycoprotein-75 (Mr = 75,000) is enriched in 4 M guanidine HCl/0.5 EDTA extracts of neonatal rat bone and growth plate tissues, whereas largely absent from heart, lung, spleen, liver, brain, and kidney. Explant cultures of these noncalcifying tissues also synthesized bone acidic glycoprotein-75 antigen, but the quantities produced were only 5% or less that obtained with calvaria. By immunohistochemistry, antigenicity is associated with the bony shaft and calcified cartilage of long bones, but is absent from associated soft tissues. These finding demonstrate that bone acidic glycoprotein-75 is antigenically distinct, predominantly localized to calcified tissues, represents a major product of normal osteoblastic cells and may undergo a characteristic fragmentation in vivo and in vitro.     

Calcium and collagen binding properties of osteopontin, bone sialoprotein, and bone acidic glycoprotein-75 from bone.

Calcium binding properties of bone acidic glycoprotein-75, osteopontin, and bone sialoprotein were determined in 10 mM imidazole buffer (pH 6.8), containing either 60 mM KCl or 150 mM NaCl. Proteins assayed were first bound to nitrocellulose to mimic substrate-bound forms in vivo; retention of phosphoproteins was determined through use of radioiodinated tracers. Binding studies were carried out both as a function of calcium concentration and the amount of phosphoprotein. In the presence of 60 mM KCl, bone acidic glycoprotein-75 exhibited the largest calcium binding capacity (139 atoms/molecule at saturation), with bone sialoprotein intermediary (83 atoms/molecule) and osteopontin lowest (50 atoms/molecule). Sites detected for each phosphoprotein exhibited overall binding constants in the 0.5-1.0 mM extracellular range. In 150 mM NaCl and 1-2 mM total calcium, phosphoproteins bound between 72 and 19 mol of calcium/mol with the same relative order. Binding was proportional to amount of phosphoprotein in either salt condition. The presence of 5 mM calcium had a different effect on concentration-dependent binding to type I collagen for each phosphoprotein. Bone acidic glycoprotein-75 alone was found to undergo an unusual calcium-enhanced polymerization reaction, confirmed by light scattering measurements, wherein collagen binding was greatest with polymeric forms. These findings demonstrate that acidic phosphoproteins from bone bind calcium atoms with a range of capacities. Calcium appears to induce conformational changes in bone acidic glycoprotein-75 which influences its self-association and binding to different substrata.     

Bone acidic glycoprotein-75 self-associates to form macromolecular complexes in vitro and in vivo with the potential to sequester phosphate ions

Monoclonal antibody HTP IV-#1 specifically recognizes a complexation-dependent neoepitope on bone acidic glycoprotein-75 (BAG-75) and a Mr = 50 kDa fragment. Complexes of BAG-75 exist in situ, as shown by immunofluorescent staining of the primary spongiosa of rat tibial metaphysis and osteosarcoma cell micromass cultures with monoclonal antibody HTP IV-#1. Incorporation of BAG-75 into complexes by newborn growth plate and calvarial tissues was confirmed with a second, anti-BAG-75 peptide antibody (#503). Newly synthesized BAG-75 immunoprecipitated from mineralizing explant cultures of bone was present entirely in large macromolecular complexes, while immunoprecipitates from monolayer cultures of osteoblastic cells were previously shown to contain only monomeric Mr = 75 kDa BAG-75 and a 50 kDa fragment. Purified BAG-75 self-associated in vitro to form large spherical aggregate structures composed of a meshwork of 10 nm diameter fibrils. These structures have the capacity to sequester large amounts of phosphate ions as evidenced by X-ray microanalysis and by the fact that purified BAG-75 preparations, even after extensive dialysis against water, retained phosphate ions in concentrations more than 1,000-fold higher than can be accounted for by exchange calculations or by electrostatic binding. The ultrastructural distribution of immunogold-labeled BAG-75 in the primary spongiosa underlying the rat growth plate is distinct from that for other acidic phosphoproteins, osteopontin and bone sialoprotein. We conclude that BAG-75 self-associates in vitro and in vivo into microfibrillar complexes which are specifically recognized by monoclonal antibody HTP IV-#1. This propensity to self-associate into macromolecular complexes is not shared with acidic phosphoproteins osteopontin and bone sialoprotein. We hypothesize that an extracellular electronegative network of macromolecular BAG-75 complexes could serve an organizational role in forming bone or as a barrier restricting local diffusion of phosphate ions. J. Cell. Biochem. 64:547–564. © 1997 Wiley-Liss, Inc.     

Characterization of fetal porcine bone sialoproteins, secreted phosphoprotein I (SPPI, osteopontin), bone sialoprotein, and a 23-kDa glycoprotein. Demonstration that the 23-kDa glycoprotein is derived from the carboxyl terminus of SPPI

Demineralizing extracts of porcine bone contain two large 66-80-kDa sialoproteins and smaller 20- and 23-kDa glycoproteins with similar chemical properties. Each protein was characterized following extraction from fetal calvariae and purification under dissociative conditions using Sepharose CL-6B, followed by fast protein liquid chromatography fractionation on hydroxyapatite and Mono Q resins. Unlike the large sialoproteins, the 20- and 23-kDa glycoproteins did not contain sialic acid. Nevertheless, affinity-purified antibodies raised against the 23-kDa protein recognized both the 20-kDa protein and a 67-kDa sialoprotein on immunoblots. These antibodies also immunoprecipitated a 60-kDa [35S]methionine-labeled protein produced by cell-free synthesis of calvarial bone mRNA, indicating that the smaller proteins were derived from the 67-kDa protein. The two sialoproteins were shown by primary sequence analysis to be secreted phosphoprotein I (SPPI, osteopontin, bone sialoprotein I) and bone sialoprotein (BSP, bone sialoprotein II). The SPPI was also characterized by its susceptibility to thrombin which produced a 23-kDa fragment, similar to the glycoprotein isolated, and a 30-kDa fragment. Amino-terminal sequence analysis of the 23- and 20-kDa proteins revealed that these proteins were derived from the carboxyl-terminal half of the SPPI molecule, the proteins showing 58% identity with human and rat, and 50% identity with mouse, SPPI sequences. Both the 23- and 20-kDa proteins appeared to be generated by the activity of an endogenous trypsin-like protease that cleaves at Arg-Ser (residues 155-156) and Lys-Ala (residues 182-183) bonds. Radiolabeling studies performed in vitro showed that the 23-kDa fragment was detectable in mineralized tissue within 4 h. The fragment was phosphorylated but, unlike SPPI, was not sulfated. The rapid generation of the 23-kDa glycoprotein and its presence in different bone tissues at different developmental stages indicate that the fragmentation of SPPI is important in bone formation and remodeling.     

Extracellular bone acidic glycoprotein-75 defines condensed mesenchyme regions to be mineralized and localizes with bone sialoprotein during intramembranous bone formation

Bone acidic glycoprotein-75 is expressed very early during in vivo models of intramembranous bone formation, highly enriched in condensing osteogenic mesenchyme after marrow ablation and the osteoprogenitor layer of tibial periosteum. Bone sialoprotein accumulates within bone acidic glycoprotein-75-enriched matrix areas at a later stage in both models. Decalcification of initial sites of mineralization consistently revealed focal immunostaining for bone acidic glycoprotein-75 underneath these sites suggesting that mineralization occurs within bone acidic glycoprotein-75-enriched matrix areas. Ultrastructural immunolocalization of bone acidic glycoprotein-75 does not support a direct association with banded collagen fibrils, but rather suggests it is a component of a separate, amorphous scaffold occupying interfibrillar spaces. Double immunogold labeling demonstrated that a sizeable proportion of bone sialoprotein particles were located within a 50-nm radius of bone acidic glycoprotein-75. These results define bone acidic glycoprotein-75 as the earliest bone-restricted, extracellular marker of osteogenic mesenchyme. Based on this early bone-restricted expression pattern and a previously documented propensity of bone acidic glycoprotein-75 to form supramolecular complexes through self-association, bone acidic glycoprotein-75 may serve a key structural role in setting boundary limits of condensing osteogenic mesenchyme.     

Identification of the type I collagen-binding domain of bone sialoprotein and characterization of the mechanism of interaction

Bone sialoprotein (BSP) is an anionic phosphorylated glycoprotein that is expressed almost exclusively in mineralized tissues and has been shown to be a potent nucleator of hydroxyapatite formation. The binding of BSP to collagen is thought to be important for the initiation of bone mineralization and in the adhesion of bone cells to the mineralized matrix. Using a solid phase assay, we have investigated the interaction between BSP and collagen. Initial studies showed that raising the ionic strength, decreasing the pH below 7, or introducing divalent cations diminishes but does not abolish the binding of BSP to collagen, indicating that the interaction is only partly electrostatic in nature. Both bone-extracted and recombinant (r)BSP exhibited similar binding affinities, indicating that post-translational modifications are not critical for binding. To identify the collagen-binding domain, recombinant peptides of BSP were studied. Peptide rBSP-(1-100) binds to type I collagen with an affinity similar to that of full-length rBSP, whereas peptides containing the sequences 99-201 or 200-301 do not bind. Further studies showed that rBSP-(1-75) competitively inhibits the binding of rBSP-(1-100), whereas rBSP-(21-100) inhibits binding to a lesser extent, and rBSP-(43-100) does not inhibit binding. These results suggest that the collagen-binding site of rat BSP is within the sequence 21-42, with residues N-terminal of this region likely also involved. This site was confirmed by the demonstration of collagen-binding activity of a synthetic peptide corresponding to residues 19-46. The collagen-binding domain, which is highly conserved among species, is enriched in hydrophobic residues and lacks acidic residues. We conclude that residues 19-46 of BSP represent a novel collagen-binding site.     

Bone acidic glycoprotein 75 inhibits resorption activity of isolated rat and chicken osteoclasts.

Matrix protein effects on the differentiated activity of osteoclasts were examined in order to understand the functional significance of bone protein interactions with osteoclasts. Bone acidic glycoprotein 75 (BAG 75) from rat calvariae inhibited the resorption of bone by isolated rat osteoclasts with IC50 = 1 nM compared to IC50 = 10 nM for chicken osteoclasts. By contrast, other phosphoproteins similarly isolated from bone were less effective in inhibiting resorption with IC50 = 100 nM osteopontin and IC50 greater than 100 nM bone sialoprotein. Likewise, RGD-containing matrix proteins vitronectin, thrombospondin, and fibronectin all displayed IC50 greater than or equal to 100 nM. Mechanistically, 10 nM BAG 75 marginally slowed, but did not block, the association of bone particles with chicken osteoclasts compared with osteopontin or control media. Pretreatment of osteoclasts with 50 nM BAG 75 had no effect on subsequent bone resorption; however, pretreatment of bone with BAG 75 before incubation with osteoclasts reduced the extent of resorption by 55%. These data suggest that a BAG 75/bone surface complex, rather than BAG 75 alone, represents the inhibitory form. Consistent with this hypothesis, direct binding studies provided no evidence of specific, high-affinity receptors on osteoclasts for BAG 75, nor was an excess of BAG 75 (100 nM) able to compete with 0.3 nM sechistatin for osteoclastic avB3-like receptors. However, BAG 75 displayed cooperative binding to tissue fragments and bone particles at concentrations greater than 10 nM, suggesting that BAG 75 self-associates into higher-order species on bone surfaces. Electron microscopy confirmed the time-dependent polymerization of BAG 75 into interconnecting filaments. These data suggest a novel, inhibitory activity for surface-bound BAG 75 on bone resorption that does not appear to involve the osteoclastic avB3-like integrin.     

Purification of a human milk protein closely similar to tumor-secreted phosphoproteins and osteopontin

A wide variety of rodent and human tumor cells secrete antigenically related phosphoproteins with molecular weights (Mr) of approximately 58,000 (hamster), 62,000 (rat, mouse), 67,000 (human) (Senger, D.R. and Perruzzi, C.A. (1985) Cancer Res. 45, 5818-5823). Expression of these phosphoproteins is transformation-related; tumor cells produce at least 10-fold or more of this protein as compared to their normal or untransformed counterparts. N-terminal and internal sequences derived from the rat tumor-secreted phosphoprotein indicate that it is identical to rat osteopontin, a bone protein with an Arg-Gly-Asp cell-binding sequence (Oldberg, A., Franzen, A. and Heinegard, D. (1986) Proc. Natl. Acad. Sci. USA 83, 8819-8823). Antibody raised to the Mr 62,000 rat tumor-secreted phosphoprotein was found to bind Mr 75,000 and Mr 35,000 components of human milk, indicating that milk contains antigenically related proteins. The Mr 75,000 protein, which is present in human milk at concentrations ranging from 3 to 10 micrograms/ml, has been purified to homogeneity. The Mr 35,000 component is apparently derived from the Mr 75,000 protein by proteolytic cleavage, and this cleavage also occurs in vitro in the presence of thrombin. N-terminal and internal amino acid sequences were derived from the Mr 75,000 milk protein and found to be similar (12/21 residues) to N-terminal and internal sequences derived from the rat tumor-secreted phosphoprotein and osteopontin. Moreover, sequence derived from the N-terminus of the human milk protein is identical to that of human bone sialoprotein I (the likely human homolog of rat osteopontin) (Fisher, L.W., Hawkins, G.R., Tuross, N. and Termine, J.D. (1987) J. Biol. Chem. 262, 9702-9708).     

Bone sialoprotein II synthesized by cultured osteoblasts contains tyrosine sulfate

Isolated mouse osteoblasts that retain their osteogenic activity in culture were incubated with [35S] sulfate. Two radiolabeled proteins, in addition to proteoglycans, were extracted from the calcified matrix of osteoblast cultures. All the sulfate label in both proteins was in the form of tyrosine sulfate as assessed by amino acid analysis and thin layer chromatography following alkaline hydrolysis. The elution behavior on DEAE-Sephacel of the major sulfated protein and the apparent Mr on sodium dodecyl sulfate gels were characteristic of bone sialoprotein II extracted from rat. This protein was shown to cross-react with an antiserum raised against bovine bone sialoprotein II, indicating that bone sialoprotein II synthesized by cultured mouse osteoblasts is a tyrosine-sulfated protein. The minor sulfated protein was tentatively identified as bone sialoprotein I or osteopontin based on its elution properties on DEAE-Sephacel and anomalous behavior on sodium dodecyl sulfate gels similar to those reported for rat bone sialoprotein I.     

Comparison of two phosphoproteins in chicken bone and their similarities to the mammalian bone proteins, osteopontin and bone sialoprotein II.

Two phosphorylated proteins of approximately 66 kDa and approximately 60 kDa mass with different DEAE-Sephacel elution patterns were isolated from chicken bone and were shown to be genetically distinct by both biochemical and immunological analysis. A tryptic peptide from the 60 kDa protein was identified that was similar to a sequence of the rat bone sialoprotein II. Both proteins showed RGD inhibited cell-attachment with the MG-63 osteosarcoma cell, and the approximately 66 kDa phosphoprotein appeared to promote cell adhesion better than human vitronectin. The two phosphoproteins appear to share functional and biochemical characteristics and to be homologous to the mammalian bone phosphoproteins, osteopontin and bone sialoprotein II.     

Purification, characterization, and studies on biosynthesis of a 59-kDa bone sialic acid-containing protein (BSP) from rat mandible using a monoclonal antibody. Evidence that 59-kDa BSP may be the rat counterpart of human alpha 2-HS glycoprotein and is synthesized by both hepatocytes and osteoblasts.

A monoclonal antibody was raised against a mineralized tissue-specific sialoprotein containing no phosphorus using partially purified noncollagenous bone matrix proteins from rats as antigen. Then the sialoprotein was purified by high performance liquid chromatography from rat mandibulae using the monoclonal antibody as a marker. The sialoprotein (59-kDa bone sialoprotein (BSP)) with a molecular weight of 59,000 contained 1.4% sialic acid but no detectable phosphorus. Immunohistochemical studies with the antibody showed that the protein was specific to mineralized tissues such as bone and dentin, and present in osteoblasts, osteocytes, and bone matrix. No other soft tissues, such as the cartilage, liver, kidney, and periosteum, were stained. However, Western blot analysis showed that plasma contained immunoreactive 59-kDa BSP. The quantitative amino acid composition of 59-kDa BSP resembled that of human alpha 2-HS glycoprotein (alpha 2-HSG) (Lee, C.-C., Bowman, B.H., and Yang, F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4403-4407; Kellermann, J., Haupt, H., Auerswald, E.-A., and Muller-Esterl, W. (1989) J. Biol. Chem. 264, 14121-14128) and rat 64-kDa protein (Franzén, A., and Heineg?rd, D. (1985) in The Chemistry and Biology of Mineralized Tissues (Butler, W.T., ed), p. 132, EBSCO Media, Birmingham, AL). Amino acid sequence analyses of the amino-terminal region and four peptide fragments of 59-kDa BSP revealed that about 50% of the amino acids were homologous with those of human alpha 2-HSG, which is known to be synthesized by the liver, transported in the bloodstream, and incorporated into calcified tissues. But when newborn rat calvaria, primary cultures of osteoblast-rich cells, and adult rat hepatocytes were incubated with radioactive leucine, immunoreactive 59-kDa BSP was detected in their conditioned medium by fluorography. Several characteristics, including the amino acid sequence, suggest that 59-kDa BSP may be the rat counterpart of human alpha 2-HSG. However, rat 59-kDa BSP is a single peptide and synthesized by both osteoblasts and hepatocytes, whereas human alpha 2-HSG is known to be a heterodimer and to be synthesized by the liver.     

Immunohistochemical localization of bone sialoprotein in foetal porcine bone tissues: comparisons with secreted phosphoprotein 1 (SPP-1, osteopontin) and SPARC (osteonectin)

Summary Bone sialoprotein (BSP) is a prominent component of bone tissues that is expressed by differentiated osteoblastic cells. Affinity-purified antibodies to BSP were prepared and used in combination with biotin-conjugated peroxidase-labeled second antibodies to demonstrate the distribution of this protein in sections of demineralized foetal porcine tibia and calvarial bone. Staining for BSP was observed in the matrix of mineralized bone and also in the mineralized cartilage and associated cells of the epiphysis, but was not observed in the hypertrophic zone nor in any of the soft tissues including the periosteum. In comparison, SPP-1 (osteopontin) and SPARC (osteonectin), which are also major proteins in porcine bone, were observed in the cartilage as well as in the mineralized bone matrix, In addition, SPARC was also present in soft connective tissues. Although SPP-1 distribution was more restricted than SPARC, hypertrophic chondrocytes, periosteal cells and some stromal cells in the bone marrow spaces were stained in addition to osteoblastic cells. The variations in the distribution and cellular expression of BSP, SPARC and SPP-1 in bone and mineralizing cartilage indicate these proteins perform different functions in the formation and remodelling of mineralized connective tissues.     

Prokaryotic expression of bone sialoprotein and identification of casein kinase II phosphorylation sites

Bone sialoprotein is an extracellular noncollagenous acidic protein that plays a role in bone mineralization and remodeling. Its expression is restricted to mineralized tissues and is subjected to variety of posttranslational modifications including phosphorylation and glycosylation. We have expressed the full-length and half domains of bovine bone sialoprotein in a prokaryotic system and identified the phosphorylation sites of casein kinase II. The N-terminal automated solid-phase sequencing defined four phosphorylated peptides: residues 28-38 (LEDS(P)EENGVFK), 51-86 (FYPELKRFAVQSSS(P)DS(P)S(P)EENGNGDS(P)S(P)EEEEEEEETS(P)), 151-165 (EDES(P)DEEEEEEEEEE), and 295-305 (GRGYDS(P)YDGQD). Nine phosphoserines were identified within the four peptides. Seven of them were in the N-terminus (S31, S64, S66, S67, S75, S76, and S86) and two were in the C-terminus (S154 and S300) of the protein.     

Further purification and characterization of newly synthesized anionic glycoconjugates secreted by cultured UMR-106 cells: evidence that the major anionic glycoconjugate secreted by these cells is similar to bone sialoprotein II.

Following incubation of UMR-106 cells for 48 h in the presence of [3H]glucosamine and [35S]sulfate, the newly synthesized anionic glycoconjugates were isolated from the culture medium by cetylpyridinium chloride/ethanol precipitation and further separated by DEAE-Sephacel chromatography into two radiolabelled fractions, a major component, UM I, and a minor component, UM II. UM I appeared to be homogeneous as shown by Sepharose CL-4B chromatography under dissociative conditions, and SDS-polyacrylamide gel electrophoresis. It showed a molecular mass of approximately 93 kDa on 4-15% gels. UM I was partially degraded by brief treatment with trypsin, releasing a small, terminal peptide that contained 47.6% of 35S but no 3H. Treatment of UM I with neuraminidase and 0.1 N H2SO4 (1 h at 80 degrees C), respectively, released 27% 3H and 38.4% 3H plus 41% 35S, suggesting the presence of a significant number of sialic acid residues, as shown by Sephadex G-50 chromatography of the digests. Amino acid analysis showed that the UM I glycoconjugate was rich in acidic amino acids (12.6% aspartic acid and 21.2% glutamic acid residues) and its N-terminal sequence was Phe-Ser-Met-Lys-Asn-Phe-, which is identical to the published N-terminal amino acid sequence of rat bone sialoprotein II. Keratanase treatment of UM I released 26% of the incorporated radioactivity, suggesting the presence of keratan sulfate chains. UM II contained a chondroitinase ABC-sensitive proteoglycan.     

Etidronate (HEBP) promotes osteoblast differentiation and wound closure in rat calvaria

The mechanisms that regulate the migration, proliferation and differentiation of osteogenic cell populations in vivo are poorly understood. Elucidation of these mechanisms is essential for an understanding of the basic processes that determine mineralized connective tissue homeostasis and regeneration. Bisphosphonates are known to regulate bone metabolism, in part through effects on osteoclastic resorption. Given previous data from other in vitro and in vivo investigations, we considered that they could also affect the proliferation and differentiation of osteoblasts in vivo. We tested this hypothesis using a bisphosphonate (ethane-1-hydroxy-1,1-bisphosphonate, HEBP, etidronate) and a calvarial wound model in which osteogenic differentiation and bone formation are coordinately induced by the wounding stimulus. Wounds through the calvarial bone were created in 20 male Wistar rats. After surgery, animals were treated every day for 1 or 2 weeks with HEBP or saline (controls) and five rats in each group were killed at 1 or 2 weeks following surgery. Cellular proliferation and clonal growth were assessed by 3H-thymidine labeling 1 h before death followed by radioautography. Cellular differentiation of osteogenic cell populations was determined by immunohistochemical staining for osteopontin and bone sialoprotein. Von Kossa and toluidine blue staining were used for the assessment of mineralization and osteoid formation, and for morphometric analysis of wound closure. At 1 and 2 weeks after surgery HEBP promoted wound closure (> twofold greater than controls, P < 0.001) and mineralized/osteoid tissue formation in the bony compartment of the wound (> 50% higher than saline controls, P < 0.05). In HEBP-treated animals there was a > 50% increase in intracellular staining for osteopontin in the endosteum-lined spaces adjacent to the wound (P < 0.05) and increased staining for osteopontin in the nascent bone at the wound margin (> 50% greater than controls, P < 0.05). However, there were reduced cell counts and labeling indices at stromal precursor sites (65% reduction compared to controls; P < 0.01). As HEBP increased osteopontin expression and osteoid/mineralized tissue formation but reduced the proliferation of precursor cells, we conclude that in addition to blockade of bone resorption and mineralization, this drug, at doses which also reversibly inhibit mineralization, may promote osteoblast differentiation as well.     

The Isolation and Characterization of Glycosylated Phosphoproteins from Herring Fish Bones

Past studies of bone extracellular matrix phosphoproteins such as osteopontin and bone sialoprotein have yielded important biological information regarding their role in calcification and the regulation of cellular activity. Most of these studies have been limited to proteins extracted from mammalian and avian vertebrates and nonvertebrates. The present work describes the isolation and purification of two major highly glycosylated and phosphorylated extracellular matrix proteins of 70 and 22 kDa from herring fish bones. The 70-kDa phosphoprotein has some characteristics of osteopontin with respect to amino acid composition and susceptibility to thrombin cleavage. Unlike osteopontin, however, it was found to contain high levels of sialic acid similar to bone sialoprotein. The 22-kDa protein has very different properties such as very high content of phosphoserine (∼270 Ser(P) residues/1000 amino acid residues), Ala, and Asx residues. The N-terminal amino acid sequence analysis of both the 70-kDa (NPIMA(M)ETTS(M)DSKVNPLL) and the 22-kDa (NQDMAMEASSDPEAA) fish phosphoproteins indicate that these unique amino acid sequences are unlike any published in protein databases. An enzyme-linked immunosorbent assay revealed that the 70-kDa phosphoprotein was present principally in bone and in calcified scales, whereas the 22-kDa phosphoprotein was detected only in bone. Immunohistological analysis revealed diffusely positive immunostaining for both the 70- and 22-kDa phosphoproteins throughout the matrix of the bone. Overall, this work adds additional support to the concept that the mechanism of biological calcification has common evolutionary and fundamental bases throughout vertebrate species.     

Osteoblast-related gene expression of bone marrow cells during the osteoblastic differentiation induced by type I collagen

Bone marrow contains multipotent cells that differentiate into fibroblasts, adipocytes, and osteoblasts. Recently we found that type I collagen matrix induced the osteoblastic differentiation of bone marrow cells. Three weeks after cells were cultured with type I collagen, they formed mineralized tissues. In this study, we investigated the expression of osteoblast-related genes (alkaline phosphatase, osteocalcin, bone sialoprotein, osteopontin, and cbfa-1) during the osteoblastic differentiation. The expression of alkaline phosphatase and osteopontin genes increased time-dependently during the osteoblastic differentiation. Osteocalcin and bone sialoprotein genes were expressed in cells that formed mineralized tissues, and both were expressed only after cells reached the mineralized tissue-formation stage. On the other hand, the cbfa-1 gene was expressed from the early differentiation stage. The Asp-Gly-Glu-Ala (DGEA) amino acid domain of type I collagen interacts with the alpha2beta1 integrin receptor on the cell membrane and mediates extracellular signals into cells. When the collagen-integrin interaction was interrupted by the addition of DGEA peptide to the culture, the expression of osteoblastic phenotypes of bone marrow cells was inhibited. These findings imply that the collagen-alpha2beta1 integrin interaction is an important signal for the osteoblastic differentiation of bone marrow cells.     

Temporal and spatial gene expression of major bone extracellular matrix molecules during embryonic mandibular osteogenesis in rats

It is not known how gene expression of bone extracellular matrix molecules is controlled temporally and spatially, or how it is related with morphological differentiation of osteoblasts during embryonic osteogenesis in vivo. The present study was designed to examine gene expressions of type I collagen, osteonectin, bone sialoprotein, osteopontin, and osteocalcin during mandibular osteogenesis using in situ hybridization. Wistar rat embryos 13–20 days post coitum were used. The condensation of mesenchymal cells was formed in 14-day rat embryonic mandibles and expressed genes of pro-(I) collagen, osteonectin, bone sialoprotein and osteopontin. Cuboidal osteoblasts surrounding the uncalcified bone matrix were seen as early as in 15-day embryonic mandibles, while flat osteoblasts lining the surface of the calcified bone were seen from 16-day embryonic mandibles. Cuboidal osteoblasts expressed pro-1(I) collagen, osteonectin and bone sialoprotein intensely but osteopontin very weakly. In contrast, flat osteoblasts expressed osteopontin very strongly. Osteocytes expressed the extracellular matrix molecules actively, in particular, osteopontin. The present study demonstrated the distinct gene expression pattern of type I collagen, osteonectin, bone sialoprotein, osteopontin and osteocalcin during embryonic mandibular osteogenesis in vivo.     

Osteonectin is a minor component of mineralized connective tissues in rat

Osteonectin is a major glycoprotein of porcine and bovine bones and teeth that is found associated with hydroxylapatite crystal surfaces. From the ability of osteonectin to bind calcium ions, it has been proposed as a possible nucleator of hydroxylapatite crystal formation. Analysis of hydroxylapatite-bound proteins of rat bone and dentine, however, has revealed that osteonectin represents only 2.5 +/- 1.5% of the hydroxylapatite-bound protein in long bones, 0.9 +/- 0.5% in calvariae, and less than 0.1% in incisor dentine of animals of different ages. Further, in vivo pulse-chase studies carried out in young adult rats have shown osteonectin to be synthesized at low levels in these tissues. Similarly, low levels of osteonectin were synthesized by rat calvarial cells in vitro. In contrast, fibroblastic cells from periodontal ligament and gingiva synthesized significantly greater amounts of osteonectin. These studies indicate that the low quantities of osteonectin in rat mineralized tissues are a consequence of low rates of formation rather than being due to rapid turnover. The virtual absence of osteonectin in incisor dentine correlates with the lack of peritubular dentine in rat, whereas the low osteonectin content of rat bones may reflect differences in their structure and biophysical properties compared with bones of larger mammals.     

Dual role for the latent transforming growth factor-beta binding protein in storage of latent TGF-beta in the extracellular matrix and as a structural matrix protein

The role of the latent TGF-beta binding protein (LTBP) is unclear. In cultures of fetal rat calvarial cells, which form mineralized bonelike nodules, both LTBP and the TGF-beta 1 precursor localized to large fibrillar structures in the extracellular matrix. The appearance of these fibrillar structures preceded the appearance of type I collagen fibers. Plasmin treatment abolished the fibrillar staining pattern for LTBP and released a complex containing both LTBP and TGF-beta. Antibodies and antisense oligonucleotides against LTBP inhibited the formation of mineralized bonelike nodules in long-term fetal rat calvarial cultures. Immunohistochemistry of fetal and adult rat bone confirmed a fibrillar staining pattern for LTBP in vivo. These findings, together with the known homology of LTBP to the fibrillin family of proteins, suggest a novel function for LTBP, in addition to its role in matrix storage of latent TGF-beta, as a structural matrix protein that may play a role in bone formation.