Bone Acidic Glycoprotein-75 Is a Major Synthetic Product of ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry

Vol. 265, No. 25, Issue of September 5, pp. 14956-14963.1990 Printed WI U.S.A.

and Molecular Biology, Inc.

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* (Received

Jeffrey Eugene

P. Gorski$, Lai, Bradley

David Karr,

Griffin, Geri and Michael

Dudley, Clark SolurshQ

From the Division of Molecular Siology and Biochemistry, Missouri 64110 and the SDepartment of Biology, University

Anti-peptide and anti-protein antisera were produced which both recognize bone acidic glycoprotein75 (Mr = 75,000) and an apparent fragment or biosynthetic intermediate (&& = 50,000) in calcified tissues andfor serum. A fragment-precursor relationship is suggested from the fact that closely spaced doublet polypeptides of M, = 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-l% 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 M, = 50,000 and 75,000 sized antigens. Bone acidic glycoprotein-75 (Mr = 75,000) is enriched in 4 M guanidine HC1/0.5 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 findings 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.

Bone is a vascularized

tissue composed

of a cellular

and an

* This work was supported in part by National Institutes of Health Grant 37078 and a Biomedical Research Support Group grant to the School of Basic Life Sciences, University of Missouri-Kansas City. Presented in preliminary form at the First Joint Meeting of the American Society for Bone and Mineral Research and the International Conference on Calcium Repulating Montreal, Can- Hormones, ada, September, 1989. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence and reprint request should be addressed: School of Basic Life Sciences, Rm. 109 BSB, University of Missouri-Kansas City, 5100 Rockhill Rd., Kansas City, MO 64110. Tel.: 816-276-2537; Fax: 816-276-5158.

Stanford&

School of Basic of Iowa, Iowa

Ronny

far publication,

Thomas,

Life Sciences, University City, Iowa 52242

February

Conway

of Missouri,

20, 1990)

Huang,

Kansas

City,

extracellular compartment, the latter of which predominates in terms of volume. Osteoblasts and osteoclasts are the major differentiated cells of bone. Osteoblasts represent the osteoid producing cells of bone and are derived from stromal or mesenchymal stem cells (1). Osteoid is composed predominantly of type I collagen (2), but contains smaller quantities of thrombospondin (3), proteoglycans (4), bone GLA protein (5, 6), osteopontin (2 ar, pp69, or Sppl) (7-g), matrix GLA protein (lo), bone sialoprotein (11, 12), and osteonectin (13). Most, if not all, of these noncollagenous proteins have been shown to be synthesized by osteoblast-like cells in culture. Synthesis of bone sialoprotein (14), osteopontin (14-16), and osteocalcin (17) is under glucocorticoid and/or vitamin D3 control. In addition, bone is the largest reservoir in the body of basic growth factors such as transforming growth factor beta (18). It is generally believed that acidic noncollagenous proteins of bone play a direct role in the processes of cell recruitment and mineralization which occur during coupled resorptive and formative phases of bone turnover. For example, both bone sialoprotein and osteopontin contain RGD sequences and mediate osteoblast-like cell adhesion in vitro (19-21). The size and shape of biologically formed calcium hydroxyapatite crystals are distinctly different from that of nonbiological counterparts (22). In vitro experiments suggest that acidic proteins containing /3 sheet structured polycarboxylates may act as calcium crystal nucleators (23). Later work has shown an additional requirement for a second sulfated component to concentrate/localize calcium ion to a region undergoing nucleation (24). The presence of extended stretches of consecutive Glu/Asp residues (9-10 residues in length) in osteopontin and bone sialoprotein are consistent with a role in calcium handling by bone. Pinto et al. (25) showed by in vitro calcium binding studies that extracts of the nonmineralized phase of young bone exhibit enough capacity to account for about 40% of the readily exchangeable calcium ion bound in uiuo by this same bone under near equilibrium conditions with %a’+. The ability of extracts from different ages of bone to bind calcium in vitro varied proportionately with the content of matrix glycoproteins and phosphoproteins (25). We isolated recently a new noncollagenous protein, bone acidic glycoprotein-75 or BAG-75,’ from the mineralized phase of rat calvarial tissue (26). Partial characterization revealed a distinctive N-terminal sequence, an Asx and Glx ’ The abbreviations used are: BAG-75, bone ABTS, 2,2’-azinobis(3-ethylbenzthiazolinesulfonic 1~3-cholamido~roovl)dimethvlammoniol-l-nropanesulfona~; 4-(%hydroxyethyl)-1-piper&ineethane~ul~on~c linked immunosorbent assay. Lo

14956

I

.I,

acidic alscoprotein-75; a&l);

CHAPS, 3HEPES, acid; ELISA, enzyme-

Distribution

and Synthesis of Bone Acidic Glycoprotein-75

content phate.

of 29%, and the presence of 7% (w/w) organic phosIon-exchange chromatography of 4 M guanidine HCl/ 0.5 EDTA extracts of rat, calvaria showed that BAG-75 copurifies with small bone proteoglycans, thus confirming its very acidic character. Although exhibiting nonidentical amino acid compositions, BAG-75 was found to share a limited sequence homology with osteopontin, as well as a reactivity with polyclonal anti-osteopontin antibodies (26). The purpose of the present study was to prepare antibody reagents for bone acidic glycoprotein-75, to characterize their immunological specificity, and to apply them to a determination of tissue distribution and site of synthesis. Our findings

document that BAG-75 is antigenically distinct, predominantly localized to calcified tissues, represents a major secretory product of normal osteoblast-like cells and may undergo a characteristic fragmentation in viva and in vitro. MATERIALS Isolation

AND

METHODS

of BAG-75

Calvarial tissues from frozen young adult rat skulls (Pel Freez Inc.) were harvested, cleaned of soft tissues and cartilaginous sutures, and washed five times with 0.05 M Tris acetate buffer (pH 7.4), containing 0.1 M c-amino-n-caproic acid, 0.005 M benzamidine hydrochloride, 0.001 M phenylmethylsulfonyl fluoride, 0.001 M p-hydroxymercuribenzoate, 1 mg/liter soybean trypsin inhibitor, and 5 mg/liter pepstatin (buffer A). Sequential extractions of lyophilized calvarial segments were performed with 4 M guanidine HCl containing inhibitors and then with 4 M rmanidine HC1/0.5 M EDTA containing inhibitors. Purification of GAG-75 (Mr = 75,000) was carried out as described by Gorski and Shimizu (26). Antibody

Production

New Zealand Red rabbits were purchased from a local supplier and preimmune bleeds obtained. A cysteinyl derivative of BAG-75 peptide 3-13 was synthesized by Immuno-Dynamics, Inc., La Jolla, CA and conjugated via a n-maleimidobenzoyl-N-hydroxysuccinimide ester coupling step (27) to bovine serum albumin, ovalbumin, and keyhole limpet hemocyanin. Rabbits were injected initially with an individual peptide-conjugate suspended in Freund’s complete adjuvant. Another rabbit was immunized with polyacrylamide gel slices containing Stains-All visualized bands of BAG-75 (M, = 75,000); the gel slices were broken into small pieces prior to injection in complete adjuvant. Booster injections in incomplete Freund’s adjuvant were made every 1-3 weeks until a useable antibody titer was reached. Resultant sera were assayed for reactivity with peptide 3-13 and/or BAG-75 by ELISA assays in microtiter wells coated with either an alternate peptide-conjugate (i.e. keyhole limpet hemocyanin conjugate for antibovine serum albumin-conjugate serum) or with purified protein. ELISA One hundred microliters of test antigen were added to microtiter wells in duplicate and each serially diluted across a twelve well row with phosphate-buffered saline containing 0.02% azide and adsorption of antigen allowed to proceed for 16 h at 37 “C. Analysis of each test antigen at a total of 12 different dilutions ensured that optical density values in the linear range were obtained for some dilutions of all test antigens in a given assay run, which proved particularly useful in comparative studies with column fractions. Two sets of plates were set up per run; one set was incubated with primary antibodies and a second set was incubated with preimmune rabbit serum (negative control). Several positive control wells were also included in each assay (i.e. anti-albumin antibodies with albumin protein adsorbed to plate). Wells were washed with phosphate-buffered saline containing 0.05% Tween 20 and then incubated with 100 ~1 of phosphate-buffered saline containing 1% ovalbumin for 2 h at 37 “C to block residual binding sites. Plates were then washed and wells incubated with primary antiserum or preimmune serum (generally l/ZOO to l/500 dilution) for 1 h at 37 “C. After removal of primary antiserum, wells were washed five times prior to addition of 100 ~1 of horseradish peroxidase conjugated second antibody (l/500). Following a 1-h incubation at room temperature in the dark and removal of unbound second antibody, calorimetric detection was accomplished by incubation for 30-90 min in the presence of 100 ~1 of 0.052 M sodium

phosphate buffer (pH and 0.09% hydrogen made at 405 nm with

5.2) containing 0.024 M citrate, 0.182 peroxide. Optical density measurements a microtiter plate reader. Western

M

ABTS, were

Blotting

Column Fractions-Aliquots (2 ml) of column fractions from a DEAE column separation of a total 4 M guanidine HC1/0.5 M EDTA extract of calvaria were boiled in the presence of 0.1% sodium dodecyl sulfate, dialyzed against two changes of 0.05% sodium dodecyl sulfate, and lyophilized separately. After solubilization and boiling in 8 M urea containing inhibitors and an excess of dithiothreitol, equal volumes of fractions were electrophoresed on duplicate sets of 7.5% polyacrylamide mini-gels (28) at 200 volts for 1 h at 4 “C. The first set of gels was transblotted onto nitrocellulose paper for 1 h at 100 V in cold transphor buffer containing 25 mM Tris-glycine (pH 8.3) and 20% methanol. The second set was transblotted onto positively charged nylon membranes (zetaprobe, Bio-Rad) for 2 h at 100 V. After blotting, gels were stained in Stains-All dye (29) to confirm protein transfer. Blots were processed for immunodetection as described previously (26). Molecular weight estimates of immunoreactive bands were made by reference to Coomassie Blue pre-stained globular standards co-electrophoresed and transblotted along with fractions. Neonatal Tissues-Oneto four-day-old AC1 strain rats were killed and the following tissues removed immediately and/or dissected free of soft tissues: calvaria, femoral growth plate, heart, lung, kidney, spleen, brain, and liver. Individual tissues were pooled and rinsed extensively with buffer A to remove blood. Tissues were frozen, lyophilized to dryness, weighed, and extracted for 72 h each with 4 M guanidine HCl containing inhibitors. Growth plate and calvarial tissues were next extracted with 4 M guanidine HC1/0.5 M EDTA containing inhibitors. Extracts were boiled in the presence of 0.1% (w/v) sodium dodecyl sulfate, dialyzed against 0.05% sodium dodecyl sulfate, lyophilized to dryness, rehydrated on the basis of initial dry weight (0.04 g/ml) in 8 M urea containing inhibitors and an excess of dithiothreitol, and processed for Western blotting as described above for column fractions. Growth of Osteoblast-like Cells and Immunoprecipitation of Labeled Antigens Growth and Labeling of Osteoblast-like Cells in Culture-Rat osteogenic sarcoma cells (ROS 17/2.8), the generous gift of Dr. G. A. Rodan (Merck Sharp and Dohme Research Laboratories), were routinely cultured in fresh modified Ham’s F-12 medium supplemented with 28 mM HEPES, 1.1 mM calcium chloride, 2.5 mM glutamine, 1 mM pyruvate, 13 mM sodium bicarbonate, 10% fetal bovine serum, and 1% kanamycin sulfate. Cells were subcultured by trypsinization once a week; medium was changed daily on days 3-7. Cultures were labeled with [YH]leucine (100 &i/ml) in leucine-depleted serum-free medium (0.1 mM leucine) for 24 h. Primary cultures of rat calvarial cells were grown as follows. Calvaria were removed under sterile conditions from eight 3-day-old Wistar pups. Using a microscope, the parietal plates were dissected free of sutures and periosteal/endosteal layers removed while tissues were submersed in Tyrode’s buffer. Following transfer to 60-mm culture dishes, tissues were diced into l-mm3 pieces in medium containing 10% serum; the suspension was distributed evenly across the dish surface and left undisturbed in an incubator for 2 days. Cultures were maintained in CMRL 1066 medium containing 10% fetal bovine serum; the medium was changed every second day and outgrowth cells reached confluency at about 14 days. Cells were released with 0.1% collagenase and 0.01% trypsin, the suspension filtered through a nitex 20 filter, and calvarial outgrowth cells established as micromass cultures (five dots/35-mm dish; 5 X lo4 cells/lO~1 dot) (30) in the same medium. Seven day primary cultures were labeled for 26 h in serine-depleted serum-free CMRL medium containing 0.1 mM serine and [“Hlserine (150 &i/ml). Immunoprecipitation of Labeled Antigem-After labeling, culture medium was removed, centrifuged at 100,000 x g for 1 h, and used directly for immunoprecipitation assays as described below. The cell layer fraction of labeled cultures was frozen/thawed three times in the presence 0.083 M sodium acetate buffer (pH 5.6), 10 mM EDTA, 100 mM e-amino-n-caproic acid, 6.4 mM benzamidine HCl, 5 mM tryptamine, and 2.5 mM l,lO-phenanthroline. Solid guanidine HCl and CHAPS were then added to a final concentration of 4 M and 0.5% (w/v), respectively, and culture flasks extracted with shaking for 1 h at 4 “C. The total extract was exchanged into 0.1 M sodium

Distribution


acetate buffer (pH 5.8), containing 6 M urea by dialysis. Detergents were then added directly to extracts to a final concentration of 0.1% sodium dodecyl sulfate, 0.1% Triton X-100, and 0.1% deoxycholate, respectively, prior to a second dialysis step against 0.1 M Tris acetate buffer (pH 7.5), containing 0.15 M sodium chloride, 10 mM EDTA, 40 mM e-amino-n-caproic acid, 10 mM benzamidine HCl, 0.25 mg/ml phenylmet.hylsulfonyl fluoride, 0.1% sodium dodecyl sulfate, 0.1% Triton X-100, 0.1% deoxycholate, and 0.02% azide. At this point, extracts were clarified by centrifugation at 100,000 X g for 1 h and supernatant fractions used for immunoprecipitation. Labeled media or cell extracts were first pre-cleared by incubation for 1 h with Staphylococcus aureus protein-Sepharose pre-loaded with normal serum IgC; 0.3-0.6 ml of labeled media or cell fractions were then added to microfuge tubes containing 0.3 ml of a 10% (v/v) suspension of S. aureus Sepharose loaded with specific antibodies. Assay tubes were brought to 1 ml with 0.01 M sodium phosphate buffer (DH 7.5). containing 0.15 mM sodium chloride. 0.5% Tween 20. and 0.k ovaibumin (PTO buffer), and mixed overnight at 4 ‘C: Assays were done in duplicate; nonspecific controls substitutedpreimmune serum for specific antiserum. S. aureus-Sepharose with bound antibody and labeled antigen was pelleted at 9,000 X g for 4 min. Immunoprecipitates were washed three times with PTO buffer and once with phosphate-buffered saline prior to solubilization by mixing/ boiling in 0.3 ml of 8 M urea, 0.5% sodium dodecyl sulfate containing inhibitors. Ten percent of solubilized immunoprecipitates were subjected to liquid scintillation counting. Selected immunoprecipitates were electrophoresed under reducing conditions on 3-20% linear gradient gels (28) and processed for autoradiography with X-Omat film after impregnation of gels with 2,5-diphenyloxalzole. Immunofluorescence

Microscopy

of Embryonic

Rat Limbs

Nineteen day embryonic rat forelimbs were removed surgically, frozen immediately, and cut on a cryostat into lo-pm cross-sections at the level of the radius and ulna. Following brief fixation with cold acetone, consecutive sections were rehydrated with phosphate-buffered saline and then demineralized for 30 min with phosphatebuffered saline containing 0.25 M EDTA. Specimens were incubated with a l/100 dilution of primary antibody (anti-BAG-75 protein serum, anti-BAG-75 peptide 3-13 serum, or preimmune rabbit serum). After rinsing with phosphate-buffered saline, fluoresceinconjugated goat anti-rabbit IgG antibody (l/300 dilution) was added to slides, unbound second antibody rinsed away, and sections mounted in glycerol containingp-phenylenediamine. Limb specimens were viewed with a fluorescence microscope and photomicrographs were taken at the same exposure settings to facilitate comparisons of relative fluorescent staining. RESULTS

Rabbit antibodies were raised separately against protein conjugates containing an N-terminal peptide (residues 3-13) of BAG-75, as well as against gel slices containing BAG-75 protein. The peptide sequence 3-13 was chosen because it represents a decamer, the minimum preferred size for peptide antigens (31), and comprises the most distinctive sequence among the fifteen known residues (26). Residues 3 and 13 are identical with similar positions in rat osteopontin. As demonstrated by the titration study illustrated in Fig. 1, both types of antisera recognize purified BAG-75 protein (Mr = 1.oo

3

E c 2

0.50

k d d

-t

0.00 0

125

AMOUNT FIG. protein

25 0

OF INPUT

1. Comparative ELISA 75 with anti-peptide

BAG-75,

37 5

J 50 I.0

nonogroms

titration of bone acidic and anti-protein sera.

glyco-

FIG. 2. Reactivity of purified bone proteins and calvarial extracts with anti-BAG-75 protein antiserum. A, samples were electrophoresed on 7.5% mini-gels, transblotted onto cationic nylon membranes, and binding of primary antibody assessed as described under “Materials and Methods.” Individual lanes: BAG-75 (2 X), 5 pg; BAG-75 (4 X), 20 pg; Calu G/E, guanidine HCI/EDTA extract of calvaria nreviouslv extracted with iust guanidine HCl; Calu G, initial guanidine HCI extract of calvaria; OF (osteopontin), 10 pg; SP II (bone sialoprotein), 10 pg; and PG I/II (small proteoglycans I and II), 10 pg. B, samples of purified bone acidic glycoprotein 75 were electrophoresed on mini-gels before or after long-term storage at 4 “C, gel lanes were then either processed for immunoblotting or stained with Stains-All. Lane 1, pattern before storage (stained); lane 2, pattern after storage (stained); lone 3, pattern after storage (immunoblot).

75,000) in ELISA assays. Whereas nonimmune serum gave a background response over the entire range of antigen tested, the anti-peptide and anti-protein sera gave rise to a detectable antigen-dependent reaction from 3 ng to more than 50 ng of input protein. Other results (not shown) indicated that antipeptide antibodies reacted with peptide 3-13 coupled to a nonhomologous protein conjugate and that immunoreactivity could be blocked by addition of free peptide 3-13. Anti-BAG-75 protein serum was analyzed by Western immunoblotting with a series of purified bone matrix proteins and tissue extracts in order to assessits specificity.* Fig. 2A depicts the results of blotting with 5 and 20 pg of purified BAG-75. A major M, = 75,000 band and two slightly smaller forms were observed at both levels; only trace recognition of other components occurred at the higher load. We have noted that a M, = 50,000 fragment has appeared upon prolonged storage of some preparations of purified BAG-75 (Fig. 2B). This fragment, detected weakly by Stains-All, is reactive with anti-BAG-75 protein antibodies, frequently exhibiting two closely spaced bands3 (Fig. 2B). In contrast, purified rat bone sialoprotein, osteopontin, and small bone proteoglycans I (biglycan) and II (decorin) were found be unreactive with anti-BAG-75 protein antibodies (Fig. 2A), under conditions where each is easily detected by its own antiserum (26, 12). BAG-75 antigenicity in calvarial extracts was found almost exclusively in the 4 M guanidine HC1/0.5 M EDTA extract, implying a requirement for decalcification for its release. Calvarial immunoreactivity was comprised of a major, broad band at M, = 50,000, with other bands at M, = 75,000 and at M, = 160,000 (Fig. 2A). Thus, anti-BAG-75 protein serum, although not cross-reactive with potential contaminants osteopontin, bone sialoprotein, and small proteoglycans, delineates two other sized protein bands associated with the mineralized phase of rat calvaria. In view of the recognition of several calvarial protein bands in addition to the previously isolated M, = 75,000 form of BAG-75, we next analyzed fractions from a DEAE-Sephacel separation of a total G/E extract (4 M guanidine HCl, 0.5 M *The anti-peptide 3-13 antiserum was ineffective in Western blotting experiments with crude or purified BAG-75. a Preliminary studies show that stromelysin treatment of purified BAG-75 (M, = 75,000) yields a long-lived fragment of approximately M, = 50,000 (P. Doskey, C. Frost, J. P. Gorski, T. Suzula, and H. Nagase, unpublished results).

Distribution


ACTION NUMBER

Stains All

C

Coomassle

Blue

- 50k

---PG.I

PG II-

- 75k

50k -

- 50k

40

60

60 COLUMN

40 50 60

70

80 90 100110 COLUMN

54 62

FRACTIONS

FIG. 3. Ion-exchange chromatography of total G/E extract of rat calvaria on DEAE-Sephacel. A, profiles of optical density (280 nm) and Alcian Blue dye binding (620 nm) measurements. B, ELISA analyses for bone acidic glycoprotein 75 and for osteopontin (OP) on individual column fractions. C, sodium dodecyl sulfatepolyacrylamide gel electrophoresis of selected column fractions on 7.5% mini-gels. Gels were stained with Stains-All or with Coomassie Blue dye as noted above gel lanes. Molecular weight estimates are empirical and based upon a comparison with globular protein standards co-electrophoresed with unknown. Please refer to “Materials and Methods” for description of protocols followed.

EDTA) of calvaria. In this way, immunoreactivity could be correlated directly with the elution profiles of individual calvarial proteins, whose identity could be established by independent means. For reference, the optical density profile at 280 nm is depicted in Fig. 3A along with that for Alcian Blue dye binding by acidic macromolecules. The gel electrophoretic pattern of every 10th fraction is presented in Fig. 3C. Only components present in fractions after 60 exhibited appreciable binding of cationic dyes; osteopontin (M, = 56,000), bone sialoprotein (iVr = 72,000), and proteoglycans I and II (iVr = 130,000 and 250,000) were detected in fractions 70-80, 80, and 90-100, respectively. Binding of anti-BAG-75 protein antibodies was monitored both by ELISA and by Western blotting on selected fractions; three major peaks of immunoreactivity were observed at fractions 54,62, and 924 (Fig. 3B). Minor immune recognition occurred at fractions 46, 68, and 74, each of which represents a major peak of protein elution (A 280 nm). In view of the potential cross-reactivity of osteopontin with anti-BAG-75 antibodies due to a limited sequence homology, we also analyzed fractions with monoclonal antiosteopontin antibodies.5 However, a single, separate peak of immunoreactivity was obtained at fraction 64. A lack of direct 4 ELISA assays munization with similar results (C. ’ A. Franzen, J.

with two monoclonal antibodies produced by imA4, = 75,000 form of BAG-75 gave qualitatively Frost and J. P. Gorski, unpublished results). P. Gorski, and M. Solursh, unpublished results.

70

80

90

100 110

FRACTlONS

FIG. 4. Comparison of immunoblotting results obtained with DEAE-Sephacel column fractions and either cationic nylon or with nitrocellulose membranes. Selected column fractions from Fig. 3 were electrophoresed on 7.5% mini-gels, transblotted, and antigens visualized as described under “Materials and Methods.” A, immunoblotting with cationic nylon membrane and with anti-BAG-75 protein antiserum. B, immunoblotting with nitrocellulose membrane and with anti-BAG-75 protein antiserum. C, negative control blot with cationic nylon membrane and with preimmune serum.

correspondence of the osteopontin and BAG-75 ELISA profiles further supports a lack of immunological relatedness between these glycoproteins. Results of Western blotting with column fractions on cationic nylon and nitrocellulose membranes are presented in Fig. 4, A and B; a negative control with normal rabbit serum is shown in Fig. 4C. Comparison of results with these membranes yields several interesting findings. First, the M, = 75,000 form of BAG-75 can be detected only with positively charged membranes and is restricted to fractions 70-100, the co-elution position for small bone proteoglycans and for BAG756 (Fig. 3C) (26). Second, anti-BAG-75 protein antibodies recognize two closely spaced bands of approximately it& = 50,000 in fractions 40-60 and 80-100. The presence of a M, = 50,000 immunoreactive band in fractions 80-100 is believed to have been produced by cleavage of BAG-75 (Mr = 75,000) prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent to DEAE chromatography. Reference to Fig. 3C indicates that one and two polypeptides of M, = 50,000 represent the predominant protein species in fractions 54 and 62, respectively; only the smaller of the doublet bands transferred to cationic nylon membranes (Fig. 4A). These findings (Fig. 4, A and B) demonstrate that the major stained polypeptide components in fractions 54 and 62 exhibit immunoreactivity with anti-BAG-75 protein antibodies. 6 Final purification of BAG-75 to homogeneity on demonstrated that the immunoreactive M, = 75,000 tical with BAG-75 protein; the immunoreactive M, was found to elute earlier and immediately following teoglycans under these conditions (C. Frost and J. P. lished results).

hydroxyapatite band was iden= 50,000 band the small proGorski, unpub-

14960

Distribution

and Synthesis of Bone Acidic Glycoprotein-75 TABLE

I

Immunoprecipitation of BAG-75 and osteopontin from radiolabeled osteosarcoma and caluarial cell cultures Radioactivity values of immune pellets were corrected for background as determined with nonimmune serum (40% that of specific immune pellets or less). Values listed are the average of duplicate assays with one cell preparation; assays were carried out with at least three separate cell preparations with similar results. Picomoles of Percent of total protein/cell Antigen Dpm/pellet Cell fraction Total dpm/cell fraction macromolecular counts fraction

% ROS 1712.8 BAG-75 BAG-75

cultures

Osteopontin Osteopontin Calvarial cultures’ BAG-75 Osteopontin

53,346 10,552

Cell layer Media

6,935,OOO 1,567,OOO

0.43 0.10

166” 37

NDh 176,649

Cell layer Media

ND 26,230,OOO

ND 1.64

812”

113,000 60,560

0.94 0.50

5,655 3,028

Media Media

” Calculated on the basis of 22 and 17 residues *ND, not detectable. ’ Less than 10% of immunoprecipitable counts ’ Calculated on the basis of 62 and 43 residues ROS 1712s CELL LAYER 290k-

?5k50k-

7 - -

ROS ,712 8 MEDIA -

--

--

-

-/

of BAG-75

and osteopontin.

were associated with the cell layer fraction of serine/mol of BAG-75 and osteopontin.

CALVARIAL MEDIA

--

d-

of leucine/mol

-,-290k

-75k -5Ok

FIG. 5. Gel autoradiographic analysis of immunoprecipitates from cultured osteoblastic cells. Primary cultures of rat calvarial outgrowth cells and of rat osteosarcoma (17/2.8) cells were labeled for 24-26 h with radioactive amino acid precursors, culture medium and cell layer fractions harvested separately, and cell fractions immunoprecipitated with antiserum or nonimmune serum. Immune pellets were solubilized and aliquots of each subjected to scintillation counting, as well as gel electrophoresis on 3-20% gradient gels and subsequent autoradiography. Band densities in lanes were optimized for presentation by varying exposure times; a summary of the scintillation counting results of immunoprecipitations is presented in Table I. Abbreviations used: NRS, nonimmune serum; antiBAG-75, antipeptide 3-13 antiserum; anCOP, monoclonal anti-osteopontin.

Taken together, our data suggest that anti-BAG-75 protein antibodies recognize the previously characterized M, = 75,000 form of BAG-75, as well as two smaller species of approximately M, = 50,000. The latter represent the major antigenic forms in calvarial extracts. Another immunoreactive band observed in total G/E extracts (Mr = 160,000) (Fig. 2A) was not detectable in column fraction blots suggesting it may be an aggregate of BAG-75 or n/i, = 50,000 proteins arising during processing or electrophoresis. Immunoprecipitation of Labeled Osteoblastic Cultures and Neonatal Explant Cultures with Anti-BAG-75 Peptide 3-13 Antiserum-ROS 17/2.8 cultures and primary cultures of normal calvarial outgrowth cells were labeled with amino acid precursors, and the medium and cell layer fractions were processed separately for immunoprecipitation as described under “Materials and Methods.” The method for extraction/ solubilization of cell layer fractions was adapted from that of Bumol et al. (33) and, yielded recoveries of at least 85% of

0.51d 0.39d

in these

cultures.

total macromolecular radioactivity. The outcome of immunoprecipitations is listed in Table I where BAG-75 content is compared with that for osteopontin. Assuming recovery of labeled antigens was quantitative in these assays, we estimate that BAG-75 represents 0.5% of total macromolecular [3H] leucine radioactivity in osteosarcoma cultures, with 81% of that total present within the cell layer fraction (Table I). In contrast, osteopontin was localized exclusively in the osteosarcoma medium fraction. ROS cells synthesized four times more osteopontin than BAG-75, whereas normal calvarial outgrowth cells produced slightly more BAG-75 protein than osteopontin (Table I). As illustrated in Fig. 5, BAG-75 antigenicity in osteosarcoma cell layer fractions was comprised of two protein bands of M, = 50,000 and 75,000; similar immunoprecipitations with labeled osteosarcoma medium fraction yielded a small amount of M, = 50,000 band observable upon longer exposure times (not shown). In a similar way, a M, = 75,000 band and a trace of M, = 50,000 polypeptide were specifically recovered from the medium of calvarial outgrowth cells (Fig. 5). An additional, nonspecific labeled band (Mr = 290,000) was found in all immunoprecipitates from calvarial and osteosarcoma cultures; initial studies showed that its level was considerably greater in the absence of a pre-clearing step. Finally, gel autoradiographs of immunoprecipitations with monoclonal anti-osteopontin antibodies, and osteosarcoma or calvaria (not shown) culture medium, displayed a predominant M, = 56,000-labeled band along with two apparent fragment bands (Fig. 5), consistent with the protein’s expected size (19). Biosynthesis studies with fibroblastic cells have suggested that conditions of continuous culture can cause abnormal expression of bone matrix proteins such as osteonectin (34). In order to rule out this possibility, we analyzed the capacities of eight freshly isolated neonatal tissues to synthesize BAG75 and osteopontin (Table II). Minced tissues, including calvaria, were incubated with [3H]leucine for 26 h and labeled proteins extracted prior to immunoprecipitations. Calvaria and growth plate tissues were the most prolific producers of bone acidic glycoprotein 75 (Table II). Measurable synthesis of BAG-75 was also detected in all the other tissues except spleen. Importantly, however, the amounts produced by heart, lung, liver, brain, and kidney were 20- to 500-fold less than that for calvaria. In summary, calcifying tissues in explant culture are the greatest producers of BAG-75 and osteopontin, synthesizing similar quantities of both.

Distribution TABLE

and Synthesis

of Bone Acidic

II

Comparison of capacities of different neonatal tissues to synthesize I”H]leucine-labeled bone acidic glycoprotein-75 and osteopontin Results shown are from a single biosynthesis experiment and are representative of two such studies. Neonatal tissues were dissected aseptically from 4-day-old rats, minced, and incubated for 26 h with 6 ml of serum-free F-12 medium supplemented with antibiotics, 100 #i/ml [“Hlleucine, and 0.1 mM leucine. Solid guanidine HCl and CHAPS were added to culture flasks to a final concentration of 4 M and 0.5%, respectively, and the suspension mixed for 1 h at 4 “C. The entire suspension was dialyzed against two changes of 4 M guanidineHCl containing inhibitors and insoluble cell debris then removed by centrifugation at 100,000 X g for 1 h. The supernatant fraction was then processed for radioimmunoassays as described under “Materials and Methods.” Immunoprecipitations were carried out with rabbit BAG-75 peptide 3-13 serum or monoclonal anti-osteopontin antibodies and with labeled tissue extracts. Results were corrected for nonspecific counts (obtained with preimmune serum) and then normalized on the basis of the amount of tissue protein determined by dye binding assay. Tissue source

Calvaria Growth Spleen Kidney Brain Heart Liver Lung

plate

Bone acidic alvcourotein

75

dpm/mg protein 100,200 (0.78%)’ 53,850 ND’ 2,900 1,810 2,400 178 5,040

nmol/g”

1,900 1,020 55 34 46 3 96

Osteopontin

14961

Glycoprotein-75

guanidine HCl extracts of the calcified tissues. The major antigenic species in calvarial G/E extract was centered at M, = 50,000; this band was absent from growth plate (Fig. 6). Rat serum also contained a closely spaced doublet at M, = 50,000 which bound anti-BAG-75 antibodies. Thus, antigens recognized by anti-BAG-75 protein serum were predominantly localized to calcified tissues and serum, whereas very minor levels were detectable in lung and kidney. Immunohistologic

Analysis

of

Embryonic

Limb

Tissues-

The morphologic distribution of bone acidic glycoprotein 75 antigen was examined with cross-sections of day 19 rat forelimbs at the level of the radius and ulna (Fig. 7). Consecutive frozen sections were fixed and demineralized prior to immunofluorescent microscopic analysis with anti-BAG-75 protein

nmol/g”

dpmlmg protein 114,380 126,670 ND’ 3,720 1,270 4,600 ND’ 1,890

2,810 3,110

’ Calculated on the basis of 22 and 17 residues of leucine/mol bone acidic glycoprotein-75 and osteopontin, respectively. b Percentage of total macromolecular counts. ’ ND, not detectable.

75k-

-75k

50k-

-5Ok

91 31 112 46 of

FIG. 6. Immunoblotting of rat neonatal tissue extracts with anti-bone acidic glycoprotein 75 protein antiserum. Neonatal tissues were lyophilized, weighed, and extracted with guanidine HCl containing inhibitors; calvarial and growth plate tissues were next extracted with guanidine HCl/EDTA. All extracts were processed for electrophoresis, adjusted to a constant volume per initial weight ratio, and immunoblotted onto cationic nylon membranes following gel electrophoresis. Abbreviations: GP G/E, growth plate guanidine HCl/ EDTA extract; GP G, growth plate guanidine HCl extract; Sp, spleen; Lu, lung; Ki, kidney; Br, brain; Ht, heart; and Li, liver (see also Fig. 2 legend).

Immunoblotting with Neonatal Rat Tissue Extracts and Anti-BAG-75 Protein Antibodies-Immunoblotting was car-

ried out with serum, and total extracts from neonatal rat heart, lung, liver, spleen, brain, kidney, femoral growth plate, and calvarial tissues (Fig. 6). When compared with calcified tissues on a dry weight basis, extracts of heart, lung, liver, spleen, kidney, and brain were negative, although lo- to 20fold higher loads permitted detection of a weak M, = 50,000 band from lung and kidney (not shown). Strong immunoreactivity for BAG-75 was found in 4 M guanidine HC1/0.5 M EDTA extracts of growth plate and calvaria (Fig. 6); each contained a reactive band at M, = 75,000 and a broad band at about M, = 160,000. A trace band at M, = 50,000 could also be observed with overloaded gel lanes containing 4 M

FIG. 7. Immunohistological analysis of rat embryonic forelimb cross-sections localizes BAG-75 antigen in hone and cartilage. Consecutive frozen sections of day 19 rat forelimbs were fixed, demineralized, and processed as described under “Materials and Methods.” A, immunofluorescence photomicrograph of section reacted with l/100 anti-BAG-75 protein antiserum. B, phase contrast micrograph of unstained section. C, negative control section treated with preimmune serum and processed for immunofluorescence analysis. (cc, calcified cartilage core; b, bony collar; p, periosteum; and ct, soft connective tissue.) Immunofluorescent image of each tissue section was photographed under the same conditions to facilitate comparisons. (Magnification: x 73).

14962

Distribution


anti-BAG-75 peptide 3-13 antibodies. As shown in Fig. 7, A and B, BAG-75 antigen was localized to the bony collar of the radius. Some calcified cartilage within the diaphyseal shaft stained positively, but less intensely than the surrounding shaft. In contrast, soft connective tissue in the section gave only background labeling (Fig. 7, A and B). Because anti-BAG-75 protein antiserum was shown above to recognize antigenic forms with different electrophoretic mobilities, it cannot immediately be determined whether the tissue reactivity observed is due to one or a combination of the M, = 50,000 and 75,000 species. As a negative control, a section was incubated with preimmune serum (Fig. 7C); only background staining was evident. Finally, anti-BAG-75 peptide 3-13 antibodies were also found to recognize specifically the bony collar and cartilaginous core of embryonic rat limb, whereas preimmune serum alone was without effect (not shown). or

DISCtJSSION

The results presented in this paper support the following statements. Anti-peptide 3-13 and anti-BAG-75 protein sera both recognize BAG-75 antigen (Mr = 75,000) and an apparent fragment or biosynthetic intermediate (Mr = 50,000); the latter represents the major antigenic form in bone extracts. A fragment-precursor relationship is suggested from the fact that closely spaced doublet polypeptides of M, = 50,000 could be produced by proteolysis upon long term storage of purified BAG-75 (Mr = 75,000) at 4 “C. The identity of immunoreactive M, = 50,000 protein(s) found in bone and in serum with these fragments is suggested by a similarity in size and reactivity with anti-BAG-75 protein antibodies. Second, BAG-75 (M, = 75,000) represents 0.5-l% of the total radiolabeled proteins synthesized by explant cultures of neonatal calvaria and growth plate, by calvarial outgrowth cultures, and by rat osteosarcoma cells. Third, BAG-75 antigen (n/i, = 75,000) is predominantly localized in neonates to bone and growth plate tissues, whereas largely absent from heart, lung, spleen, liver, brain, and kidney. Within long bones, BAG-75 antigenicity is associated with the bony shaft and some calcified cartilage, and absent from associated soft tissues. Consistent with a localization to the bone mineral phase, recovery of bone acidic glycoprotein-75 from neonatal and adult calcified tissues required demineralization. Fourth, presumed fragments (doublet bands of M, = 50,000) are found in serum as well as in neonatal bone, whereas absent from growth plate. These findings demonstrate that bone acidic glycoprotein-75 is antigenically distinct, predominantly localized to calcified tissues, represents a major product of osteoblastic cells and may undergo a characteristic fragmentation in vivo and in vitro. It is evident that glycoproteins found in bone could be remnants of cartilage or synthesized elsewhere and taken up by bone due to an affinity for hydroxyapatite. In vitro synthesis studies were used to resolve this question. As exemplified by the secretion of osteonectin by fibroblasts in culture but not in skin in vivo (34, 35), biosynthesis of BAG-75 by cultured osteoblast-like cells could also be the result of artifactual expression. However, combined studies with explant cultures of calvarial and growth plate tissues, and with cultured bone cells, provide clear evidence that the presence of BAG-75 in bone is due to its synthesis by resident cells and its specific retention within calcified bone matrix. The amount of BAG-75 synthesized by calvarial explants is twothirds of that for osteopontin, a major product of osteoblasts (14, 36). Production of these sialoproteins by cultured calvarial outgrowth cells and osteosarcoma cells was also within the same range, i.e. 131 and 25% for BAG-75 as compared with osteopontin. Previous in situ hybridization (37) and immu-

nological (33) surveys of osteopontin synthesis and distribution noted its presence in kidney and the inner ear. However, our biosynthetic results with neonatal tissues in explant culture show that production of osteopontin (and BAG-75) is more than 30 times greater in bone than for kidney. Our findings indicate that bone acidic glycoprotein 75 is itself a prominent translation product of bone cells. Based upon its biochemical properties, BAG-75 shares a general structural homology with osteopontin and bone sialoprotein. While little direct evidence exists regarding the function of BAG-75,7 prior efforts to establish mechanistic roles for bone sialoproteins have examined their kinetics of appearance and distribution in in vitro models of bone formation (40,41). Stein and colleagues (42) divided the process into replication, matrix maturation, and mineralization phases. In vitro, osteopontin and osteocalcin synthesis begins at the end of the matrix maturation phase; calcium deposition correlated closely with accumulation of these two proteins. Combined with histological evidence for the enrichment of acidic and sulfated groups within mineralizing nodules (43) and the ability of sulfated and polycarboxylated macromolecules to act as crystal growth regulators (24), the aforementioned findings imply a role for osteopontin, sialoprotein II, and possibly BAG-75 in mineral crystal nucleation and/or growth. Interestingly, cloned sequences of the former two proteins (19, 44) are devoid of consensus “EF hand” calcium binding motifs (45). In addition to Asp/Glu contents of 2529%, unusual poly-Asp/Glu sequences are present in osteopontin and sialoprotein II, respectively, and BAG-75 also contains a stretch of six acidic residues at the N terminus (26). Of note, the C-terminal region of muscle calsequestrin also contains stretches of up to 14 connective Asp/Glu residues (46, 47). Calsequestrin is also devoid of the EF hand motif and acts as a weak affinity, large capacity calcium binder in the junctional sarcoplasmic reticulum (48). Calcium binding by cardiac calsequestrin alters its conformation and prevents binding to junctional membrane proteins, which occurs in the presence of low calcium ion concentration. Further work is necessary to determine whether calsequestrin provides a useful analogy for calcium binding by bone sialoproteins. Disease models of bone suggest that bone matrix proteins may influence osteoclast differentiation and function. Limbs from osteopetrotic mutant tl/tl are unable to support osteoclast development and differentiation when supplied with either normal or mutant splenic osteoclastic precursor cells (49). These results may indicate that the amount or arrangement of bone matrix proteins in tl/tl bone is faulty. Horton and colleagues (50) recently characterized the osteoclast functional antigen as a member of the vitronectin receptor family of integrin cell adhesion molecules. Developmental expression of the vitronectin receptor occurs first in presumptive mononuclear osteoclast precursor cells in the outer periosteum of long bones, with progressive accumulation of receptor positive mono- and multinucleated cells at this site and within developing marrow (51). Bone sialoprotein (and apparently osteopontin) are known to mediate adhesion of osteosarcoma and fibroblastic cells via this receptor family (19, 21) and may also participate in osteoclast attachment to calcified bone matrix. Although a glycopeptide of M, = 23,000 derived from bone sialoprotein was isolated from bone by Herring and co-workers in 1967 (52), our results represent the first immunological detection of an apparent fragment of a noncollagenous protein 7 Initial experiments indicate activity with cultured osteosarcoma RGD-containing peptides (39).

that

BAG-75 cells which

exhibits cell adhesive is inhibitable by small

Distribution


within bone. The role of proteases in bone formation is not clear, although they play an obvious role in resorption of bone matrix mediated by osteoclasts. Neutral metalloproteinases such as stromelysin and collagenase are secreted by bone cells in culture (53); plasminogen activator is also detectable in bone tissue (54). Einhorn et al. (55) showed that neutral protease activity in callus tissue peaked at 14-17 days postfracture, a time which corresponds to a period of vascular invasion and initial mineral deposition (56). A positive role for neutral metalloproteinase activity in bone formation was suggested by Tyree (57), who showed that binding of osteonectin to type I collagen fibrils was enhanced following limited proteolysis. With regard to BAG-75, we assume that proteolytic cleavage in bone produces a M, = 50,000 fragment; preliminary results indicate that stromelysin is capable of generating a similar sized fragment from BAG-75 in uitro.3 Much interest is focused on the evaluation of bone-derived serum markers for use in diagnosis and in following treatment of osteoporosis patients (32, 58). Current efforts are now directed toward a definitive structural identification of the presumed M, = 50,000 fragment of BAG-75 from bone and serum. Acknowledgments-We wish to express our gratitude to Colleen Frost and Karen Jensen for their excellent technical work. Special thanks to Philistia A. Bronston for preparation of the manuscript. REFERENCES 1. Owen, M. (1985) in Bone and Mineral Research (Peck, W. A., ed) Vol. 3 pp. l-25, Elsevier Science Publishing Co., Inc., New York 2. Triffit, T. J. (1980) in Fundamental and Clinical Bone Physiology (Urist, M. R., ed) pp. 45-82, J. B. Lippincott, Philadelphia 3. Gehron Robey, P., Young, M., Fisher, L., and McClain, T. (1989) J. Cell Biol. 108, 719-727 4. Herring, G. M. (1968) Rio&em. J. 10'7, 41-49 5. Hauschka, P. V., Lian, J. B., and Gallop, P. M. (1975) Proc. Nutl. Acud. Sci. U. S. A. 72, 3925-3929 6. Price, P. A., Otsuka, A. S., Poser, J. W., Kristaponis, J., and Raman, N. (1976) Proc. Nutl. Acud. Sci. U. S. A. 73, 14471451 7. Fisher, L. W.. Whitson, S. W., Avioli, L. V., and Termine, J. D. (1983) J. Biol. Chem. 258, 12723-12727 8. Franzen. A.. and Heineeard. -,. D. (1985) Biochem. J. 232.715-724 9. Prince, C. W., Oosawa, T., Butler, W. T., Tomana, M., Bhown, A. S., Bhown, M., and Schrohenloher, R. E. (1987) J. Biol. Chem. 262,2900-2907 10. Price, P. A., and Williamson, M. K. (1985) J. Biol. Chem. 260, 14971-14975 11. Franzen, A., and Heinegard, D. (1985) in The Chemistry and Biology of Mineralized Tissues (Butler, W. T., ed.) pp. 132-141, EBSCO Media, Birmingham, AL 12. Fisher, L. W., Hawkins, G. R., Tuross, N., and Termine, J. D. (1987) J. Biol. Chem. 262,9702-9708 13. Termine, J. D., Kleinman, H. K., Whitson, W. S., Conn, K. M., McGarvey, M. L., and Martin, G. R. (1981) Cell 26,99-105 14. Oldberg, A., Jirskog-Hed, B., Axelsson, S., and Heinegard, D. (1989) J. Cell Biol. 109, 3183-3186 15. Prince, C. W., and Butler, W. T. (1987) Collagen Relat. Res. 7, 305-313 16. Yoon, K., Buenaga, R., and Rodan, G. A. (1987) Biochem. Biophys. Res. Commun. 148,1129-1136 17. Price, P. A., and Baukol, S. A. (1980) J. Biol. Chem. 255,1166011663 18. Centrella, M., McCarthy, T. L., and Canalis, E. (1988) FASEB J. 2,3066-3073 19. Oldberg, A., Franzen, A. and Heinegard, D. (1986) Proc. Nutl. Acud. Sci. U. S. A. 83,8819-8823 20. Oldberg, A., Franzen, A., Heinegird, D., Pierschbacher, M., and

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