Biomedical subjects

J P Gorski

Publications and source records attributed to J P Gorski.

At least 19 recordsLinked to original sources

PubMed2001

A comparative study of sialic acid-rich proteins in rat bone and dentin.

Four sialic acid-rich (SA-rich) proteins found in bone and dentin, osteopontin (OPN), bone sialoprotein (BSP), bone acidic glycoprotein-75 (BAG-75), and dentin matrix protein 1 (DMP1), share some common features. We used SDS-PAGE and Western immunoblots to analyze and compare SA-rich proteins in bone and dentin extracts from rats with a single chromatographic procedure. OPN was detected in dentin extracts, with a relative level less than one-seventieth of that in bone. Both bone and dentin BSP demonstrated an extremely broad distribution pattern, probably due to a high degree of heterogeneity in post-translational modifications. BAG-75 in both bone and dentin was detected as an 83 kDa band, dramatically distinct from that of DMPI. Using a polyclonal antibody raised against a purified bone 57 kDa protein (a portion of DMPI), we detected 150 kDa protein bands in bone fraction; the same bands were recognized by antirecombinant rat DMPI antibody. Bands from dentin migrating at about 150 kDa in earlier fractions and progressing to 200 kDa in later fractions showed a clear immunoreactivity to the anti-57 kDa antibody. We conclude that the majority of DMPI in rat bone is processed into fragments, whereas that in dentin remains intact.

Animals↗

PubMed2000

Hypercalcemia during the osteogenic phase after rat marrow ablation coincides with increased bone resorption assessed by the NTx marker.

Marrow ablation is a model of bone turnover in which the excavated tibial intramedullary cavity is rapidly and reproducibly filled by osteoblasts with new woven bone (days 6-8), which is then rapidly resorbed by osteoclasts (days 10-15). We showed previously (Magnuson et al., 1997) that marrow ablation induces a dramatic hypercalcemia and hypercalciuria in rats that unexpectedly peaked at the time of maximal osteogenesis and continued throughout the subsequent resorption phase. Based upon the amount of calcium mobilized and a peak of urinary hydroxyproline, we suggested that the hypercalcemia and hypercalciuria were due to increased systemic osteoclastic bone resorption induced by marrow ablation. We now apply a new enzyme-linked immunosorbent assay for rodent alpha(2)(I) N-telopeptide (NTx), a marker of bone resorption, to the marrow ablation model to demonstrate that excretion of NTx parallels that of calcium release in the operated control group. Specifically, maximal NTx/creatinine excretion coincides with the onset of hypercalcemia on days 7-8. A peak of NTx was also observed in methylprednisolone- and deflazacort-treated ablated animals. Analyses for urinary free deoxypyridinoline crosslink failed to detect a significant ablation-induced change in excretion. Interleukin 6 activity was increased in all operated control and glucocorticoid-treated groups after marrow ablation, whereas serum parathyroid hormone remained at presurgical levels in operated controls throughout the 15-day study period. The NTx results confirm that bilateral tibial marrow ablation induces a burst of extratibial bone resorption and hypercalcemia 7-8 days later. We have estimated that the osteogenic phase of the ablation model deposits 40 mg of calcium as hydroxyapatite crystals within the intramedullary cavity on days 6-8; this represents 33%-50% of the total blood calcium content of a young rat. We hypothesize that the size and rapidity of this demand for ionized calcium is met through an extratibial bone resorption pathway of osteoclast formation and activation that anticipates and fulfills this need, and that is initiated at the time of marrow ablation.

Animals↗

PubMed1999

Recombinant expression and characterization of dentin matrix protein 1.

Dentin matrix protein 1 (DMP1) is an extracellular matrix noncollagenous protein (NCP) initially isolated from dentin and now found to be present in calcified tissues like calvaria and long bone. The characteristic feature of DMP1 is that it contains a large number of acidic domains and has properties which implicate it as a key participant in regulating matrix mineralization. The level of DMP1 in the tissue is sparse and it is not easily isolated from dentin because it copurifies with other dentin NCPs. The exact function of DMP1 is not known and this is due to the inherent difficulty of obtaining enough protein from the mineralized tissues. In order to understand the physiologic role for DMP1 during the formation of mineralized tissues we have produced milligram quantities of recombinant DMP1 in E. coli. The objective of this work was: (1) to prepare unmodified apoprotein so that it could be used for studying the function of DMP1; and (2) to prepare polyclonal antibody against the recombinant DMP1 antigen. The DMP1 polyclonal antibody did not cross-react with other NCPs present in dentin or with bone acidic glycoprotein-75 (BAG-75) present in the bone matrix, confirming the specificity of this antibody and thus making it a valuable tool to determine the in vivo function of DMP1.

Amino Acid Sequence↗

PubMed1998

Mutations in extracellular matrix molecules.

Genetic studies of humans and mice continue to highlight the nonredundant mechanical role of components in complexes that anchor cells to extracellular matrices. At the same time, recent data provide exciting insights into nonredundant, critical roles of transcription factors in regulating differentiation and function of matrix-producing cells.

Epidermolysis Bullosa, Junctional↗

PubMed1998

Is all bone the same? Distinctive distributions and properties of non-collagenous matrix proteins in lamellar vs. woven bone imply the existence of different underlying osteogenic mechanisms.

The purpose of this review is to summarize recent functional and structural findings regarding non-collagenous matrix proteins in bone and teeth, to compare gene locations for bone and tooth matrix proteins with loci for hereditary skeletal diseases, and to present several provocative hypotheses which integrate this new information into a physiological context. Hypothesis I proposes that the molecular composition of rapidly deposited and mineralized woven bone, as well as the responsiveness of cells synthesizing woven bone to stimuli, is different from that for more slowly synthesized lamellar bone, implying the existence of distinctive osteogenic mechanisms. This review of recent research strongly supports this proposal. Briefly, the protein composition of woven bone matrix is enriched in acidic phosphoproteins BAG-75 and BSP, which are not expressed in lamellar bone, which is itself enriched in osteocalcin. De novo deposition and mineralization of woven bone occurs faster than in lamellar bone by means of a matrix-vesicle-assisted mechanism. Deposition of woven bone occurs at sites experiencing biomechanical strains higher than those experienced by lamellar bone. In addition, woven bone in metaphyseal regions is more susceptible to osteoclastic resorption after space flight, ovariectomy, and loss of weightbearing than is lamellar bone. Finally, osteoprogenitor cells responsive to parathyroid hormone reside in the metaphyseal region of long bones. Taken together, these findings suggest that Hypothesis I represents a useful paradigm for future studies. Specific functions mediated by most individual bone and tooth matrix proteins remain uncertain. A review of current literature suggests that the functionality of skeletal matrix proteins is expressed through specific binding sites composed of particular species-conserved structural motifs (Hypothesis 2). Examples include the previously recognized Asp-Ser-Ser motif of dentin phosphophoryns and the gamma-carboxyglutamic acid motif of matrix GLA protein and osteocalcin. A new polyacidic amino acid motif composed of consecutive Asp and Glu residues (n > 7) was defined in extracellular matrix components osteopontin, bone sialoprotein, and bone acidic glycoprotein-75 on the basis of strong functional analogies with similar polyacidic stretches in divalent metal storage proteins of the endoplasmic reticulum and sarcoplasmic reticulum. These structural motifs represent prime targets for future structure-function studies in vivo and in vitro.

1-Carboxyglutamic Acid↗

PubMed1998

Hydroxyapatite induces autolytic degradation and inactivation of matrix metalloproteinase-1 and -3.

In the course of studies to identify a protease capable of producing a long-lived 50 kDa fragment of bone acidic glycoprotein-75 (BAG-75), it was observed that incubation of matrix metalloproteinase (MMP)-3 (stromelysin 1) with preparations of BAG-75 led to inactivation of proteolytic function, e.g., an inability to fragment 125I-labeled BAG-75 added subsequently. MMP-1 (interstitial collagenase) was also inactivated by exposure to BAG-75 preparations. Investigation of the mechanism revealed that BAG-75 preparations contained millimolar levels of inorganic phosphate which formed hydroxyapatite crystals under digestion conditions. Hydroxyapatite crystals alone and in BAG-75-hydroxyapatite complexes induced the autolytic degradation of both active and precursor forms of MMP-1 and MMP-3. Autolytic degradation in the presence of hydroxyapatite was demonstrated by a loss in catalytic function assayed with peptide and/or protein substrates, and, by fragmentation into polypeptides of <10 kDa. The fate of MMP-3 incubated with hydroxyapatite depends upon the time of incubation, the free calcium concentration, and the concentration of crystals. Specifically, hydroxyapatite-induced autolysis requires a near physiological free calcium concentration of 0.5-1.0 mM. Autolysis was maximal in the presence of 150 microg/ml hydroxyapatite where MMP-3 was only partially bound to crystals. However, autolysis also occurred at higher crystal concentrations where all input MMP-3 was bound (>1000 microg/ml), suggesting that autolysis may be mediated by bound enzyme. The effect of hydroxyapatite appears to be specific for MMP-1 and MMP-3 since the catalytic activity of chymotrypsin, trypsin, papain, and thermolysin remained unchanged after exposure to hydroxyapatite. These results document for the first time a novel catalytic role for hydroxyapatite crystals in vitro and provide an initial biochemical characterization of the intermolecular, autolytic, calcium ion-dependent, matrix metalloproteinase-specific degradative mechanism.

Animals↗

PubMed1997

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.

Animals↗

PubMed1997

Bilateral tibial marrow ablation in rats induces a rapid hypercalcemia arising from extratibial bone resorption inhibitable by methylprednisolone or deflazacort.

The goals of this study were to quantitate biochemical markers of bone metabolism on days 1-15 after bilateral tibial marrow ablation surgery in young adult rats and to determine the effect of a single dose of methylprednisolone (2 mg/kg) or deflazacort (2.5 mg/kg) given at the time of ablation. Unexpectedly, serum calcium levels rose to a maximum of 15.9 mg/dl on day 7 after marrow ablation and remained above normal through day 15. This increase was blocked by a single intramedullary injection of methylprednisolone or deflazacort immediately following ablation; however, the fact that both drugs produced a characteristic rapid 3- to 10-fold increase in the serum alpha 2-macroglobulin level demonstrates that the drugs rapidly reached the circulation. Both methylprednisolone and deflazacort also inhibited intramedullary deposition of collagen by 40-60% on day 7, a time near which operated control animals achieved maximal accumulation of new bone in this model. Histological comparisons among the three experimental groups were largely consistent with biochemical results. The urinary hydroxyproline/creatine ratio for the operated control group doubled on day 3 and then returned to presurgical levels on day 7 and later. The timing and size of the hydroxyproline/creatinine peak, as well as the fact that the intratibial osteoclastic response peaks on days 8-10 after ablation, suggests it results from extratibial bone resorption induced by marrow ablation. Consistent with this rationale, urinary calcium excretion in operated controls rose 9-fold from day 0 to day 3 and appeared to plateau over the period from day 3 to day 9, before returning to a near presurgical level on day 15. Elevated excretion of calcium noted on days 9-15 in deflazacort-treated animals, which occurs in the absence of a detectable increase in resorption marker hydroxyproline, may however be due to the known action of glucocorticoids in increasing kidney filtration of calcium. In summary, this is the first report to show that bilateral tibial marrow ablation in rats causes a rapid hypercalcemia and calciuria which is accompanied initially by a peak of bone resorption marker urinary hydroxyproline. We speculate that the source of calcium and hydroxyproline is extratibial osteoclastic bone resorption induced by circulating cytokines whose release from ablated tibias or osteoclastogenic action is inhibitable by methylprednisolone and deflazacort.

Animals↗

PubMed1996

Bone acidic glycoprotein-75 self-associates to form large macromolecular complexes.

Bone acidic glycoprotein-75 (BAG-75) displays a strong propensity to self-associate to form large fibrillar complexes above concentrations of 7 x 10(-8) M; acidic phosphoproteins osteopontin and bone sialoprotein do not form similar complexes. Although the majority of the data supporting this conclusion is derived from in vitro studies, the fact that similar sized complexes are observed in crude extracts of bone and calcified cartilage suggests that macromolecular BAG-75 complexes are also a component of mineralized matrices in vivo. An awareness of the existence of complexes in extracts from bone necessitates that these forms are accounted for in terms of the relative amounts of individual acidic phosphoproteins in bone matrix. We now estimate that the amount of BAG-75 in rat calvarial bone is equivalent to that of osteopontin. While BAG-75 is capable of binding up to 139 atoms of calcium/mole with an average affinity constant of 0.5-1.0 x 10(-3) M, millimolar concentrations of calcium are not required for self-association. Assuming macromolecular diffusion within osteoid is restricted, osteoblastic cells could control the extent of self-association through the rate at which BAG-75 is synthesized and secreted into the osteoid matrix. Based on these findings, we hypothesize that BAG-75 self-associates to form fibrillar complexes in vivo which function in a supportive mechanical role and/or as an electronegative ionic barrier. Electronegative BAG-75 barrier structures could play a role in concentrating phosphate ions within bone matrix, thus facilitating mineralization.

Animals↗

PubMed1995

Conformational analyses on soluble and surface bound osteopontin.

Immunohistology of calvarial sections revealed that staining with monoclonal anti-osteopontin antibodies (clone MPIIIB10) is minimal unless sections are first treated with EDTA. In contrast, following treatment of sections with EDTA, strong staining of mineralizing osteoid areas and osteoblast-like cells was noted (Fig. 1B). Immunostaining for osteopontin appeared to be specific in that controls which substituted rabbit IgG or normal mouse ascites fluid for monoclonal antibody, or which omitted monoclonal antibody uniformly gave background results (Fig. 1C). In an effort to circumvent problems of antibody accessibility we examined the immunoreactivity of OP when adsorbed to plastic and hydroxyapatite surfaces. Although OP bound to plastic surfaces is reactive with MPIIIB10 antibodies, OP adsorbed to hydroxyapatite crystal surfaces is not recognized by these antibodies as assessed by two detection methods. These results demonstrate that most or all of OP bound to hydroxyapatite exhibits a different conformation than when bound to plastic surfaces. On the basis of immunohistologic results with calvarial sections, we suggest that the conformation of native OP in bone and of isolated OP adsorbed to hydroxyapatite may be similar. Finally, solution circular dichroism and Fourier-transformed infrared spectroscopic studies indicate that the conformation of bone OP is dependent upon its concentration, and, secondarily to the presence or absence of calcium ion. With both spectroscopic methods, addition of calcium appeared to increase the extent of disordered structure. We suggest that these findings support our hypothesis that bone matrix proteins exhibit a different conformation when adsorbed on hydroxyapatite crystal surfaces. Assumption of a more organized secondary structure in concentrated OP solutions (i.e., 15 mg/ml) is consistent with these results in that local concentrations of OP within a semisolid matrix may approach or exceed levels used here.

Animals↗

PubMed1995

Collagen metabolism and tooth eruption: the effects of sodium morrhuate infusions on premolar eruption in dogs.

To test the essential contribution to tooth eruption of the known high level of collagen metabolism in the periodontal ligament, we have infused the crypts of erupting premolars in dogs with sodium morrhuate, a compound known to reduce production, hydroxyproline content and maturation of collagen. Infusions of sodium morrhuate early or later in eruption for more than half the period of eruption had no effect on the process evaluated radiographically and clinically. These data, considered together with other studies, suggest that collagen metabolism per se plays no essential role in tooth eruption.

Animals↗

PubMed1995

The mechanisms and mediators of tooth eruption--models for developmental biologists.

Tooth eruption is a localized process in the jaws which exhibits precise timing and bilateral symmetry. It involves resorption and formation of bone on opposite sides of the erupting tooth and these activities depend on the dental follicle, a thin connective tissue investment of the developing and erupting tooth. Biochemical studies have shown that during eruption cells, proteins and enzymes change in the dental follicle and several growth factors and proteins known to accelerate or retard eruption have been identified. This review discusses these aspects of tooth eruption and proposes testable hypotheses and strategies that can make studies of tooth eruption new experimental opportunities for developmental biologists.

Animals↗

PubMed1994

The effect of removing the true dental follicle on premolar eruption in the dog.

Eruption is a highly localized process during which the bone resorption and formation that occur on opposite sides of the tooth are dependent upon the surrounding soft tissues, the true dental follicle externally and the enamel organ internally. To examine the ability of the enamel organ to cause eruption the external layer (dental follicle) was removed just prior to and up to 4 weeks before eruption in 13 mandibular premolars in dogs and eruption followed clinically, radiographically and histologically. None of the teeth without dental follicles erupted but three teeth from which the follicle was separated then replaced did erupt. These data indicate that the enamel organ without the dental follicle cannot support tooth eruption and provide indirect evidence for the central role of the dental follicle, alone or in combination with the enamel organ, in eruption.

Animals↗

PubMed1993

Glycosaminoglycans and periodontal disease: analysis of GCF by safranin O.

The purpose of this study was to quantify glycosaminoglycans (GAG) released into the gingival crevicular fluid (GCF) during health, gingivitis, and adult periodontitis. The investigation tested the hypothesis that increased amounts of GAG can be measured in GCF associated with gingivitis and adult periodontitis as compared to health. An individual patient's sampling sites were assigned to either a health (control) group or 1 of 3 experimental groups, gingivitis, periodontal "maintenance" (perio-M), or periodontal "non-maintenance" (perio-NM) according to standard clinical criteria of pocket probing depth, bleeding on probing, and radiographic evidence of bone loss. The perio-M group was defined as a periodontal patient who had received a dental prophylaxis and/or root planning within 6 months prior to GCF collection. The perio-NM group had received no periodontal therapy during the previous 6 months. Subsequent to air-drying and isolation, GCF was collected by a microcapillary pipette held at the gingival margin. All fluid samples were digested overnight at 37 degrees C with 25 micrograms of papain and analyzed for GAG content using a chondroitin-4-sulfate standard. Data generated from safranin "O" dye binding assays of GAG revealed 4.41 +/- 9.82 ng GAG from the health (control) group (n = 23); the gingivitis group (n = 13) showed 15.23 +/- 11.85 ng GAG/sample; perio-M group (n = 11) showed 23.64 +/- 12.98 ng GAG/sample and the perio-NM group (n = 12) exhibited 119.08 +/- 33.14 ng GAG/sample.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1993

A comparative analysis of subchondral replacement with polymethylmethacrylate or autogenous bone grafts in dogs.

A comparative analysis was made of subchondral replacement with polymethylmethacrylate and autogeneic bone grafts in defects in the medial femoral condyles of dogs. The defect produced a 50% reduction in subchondral stiffness. An in vitro preparation helped establish that subchondral stiffness returned to normal after reconstruction with polymethylmethacrylate. The in vivo model demonstrated a reduction in subchondral stiffness in both groups at three weeks, but the bone grafted side returned to normal and the methylmethacrylate side recovered to 79% of the control at 12 weeks. There were no deleterious effects on the articular cartilage in either group when analyzed histologically and biochemically. A marked increase in new bone formation and subchondral porosity was found in the polymethylmethacrylate groups. This study supports the clinical use of subchondral polymethylmethacrylate after the exteriorization and curettage of benign bone tumors such as giant cell tumors.

Animals↗

PubMed1992

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.

Animals↗

PubMed1992

Acidic phosphoproteins from bone matrix: a structural rationalization of their role in biomineralization.

Osteopontin, bone sialoprotein, and bone acidic glycoprotein-75 are three acidic phosphoproteins that are isolated from the mineralized phase of bone matrix, are synthesized by osteoblastic cells, and are generally restricted in their distribution to calcified tissues. Although each is a distinct gene product, these proteins share aspartic/glutamic acid contents of 30-36% and each contains multiple phosphoryl and sialyl groups. These properties, plus a strict relationship of acidic macromolecules with cell-controlled mineralization throughout nature, suggest functions in calcium binding and nucleation of calcium hydroxyapatite crystal formation. However, direct proof for such roles is still largely indirect in nature. The purpose of this review is to present two speculative hypotheses regarding acidic phosphoprotein function. The goal was to use new sequence information along with database comparisons to develop a structural rationalization of how these proteins may function in calcium handling by bone. For example, our analysis has identified a conserved polyacidic stretch in all three phosphoproteins which we propose mediates metal binding. Also, conserved motifs were identified that are analogous with those for casein kinase II phosphorylation sites and whose number correlates well with that of phosphoryl groups/protein. A two-state conformational model of calcium binding by bone matrix acidic phosphoproteins is described which incorporates these findings.

Amino Acid Sequence↗

PubMed1992

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.

Animals↗