Bone acidic glycoprotein 75 inhibits resorption ... - The FASEB Journal

Bone

acidic

isolated MASAHIW

75 inlibits isorp.tion rat and chicken ostloclasts glycoprotein

SATO,”2

WJLLL4M

P. GORSKI4 Department Pennsylvania University

GRASSER,3

SAIDY

PULLENJL(MP,4

AND JEFFREY

of Bone Biology and Osteoporosis Rescarch Merck Sharp & Dohme Research Laboratories, West 19486, USA; and Division of) oleculaiBioiogjr #{225}Id Biodiemistry, School of Basic Life Sciences, of Missouri-K.C., Kansas City, Missonri 641$, USA

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 IC30 = 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 IC30 > 100 nM bone sialoprotein. Likewise, RGD-containing matrix proteins vitronectin, thrombospondin, and fibronectin all displayed IC30 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 aB3-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 aB3-like integrin.Sato, M.; Grasser, W.; Harm, S.; Fullenkamp, C.; Gorski, J. P. Bone acidic glycoprotein 75 inhibits resorption activity of isolated rat and chicken osteoclasts. FASEBJ. 6: 2966-2976; 1992. bone

resorption

polymerization

bone

OSTEOCLASTS ARE THE MULTINUCLEATED cells responsible for the resorption of both mineral and organic components of bone. Activated osteoclasts polarize their cytoplasm and form specialized organelles called the clear zone and ruffled border, which mediate bone resorption. The clear zone consists of attachment complexes organized into a ring pattern that surround the ruffled border wherein rapid secretion of acid and proteases take place (1-5). The clear zone organelle

2966

COLLEEN

of

.

ABSTRACT

Key Words: osteoclost matrix phosphoproteins

HARM,

activity

contains a-actinin and myosin cross-linked actin linked to a membrane-spanning complex consisting lin, talin, and integrins that recognize Arg-Gly-Asp 5-containing

matrix

proteins

in bone

(6-10).

The

Point,

filaments of vincu(RGD) molecular

interactions that regulate the reorganization of the osteoclast cytoplasm to form these structures are poorly understood at present. The primary organic constituent of bone is collagen, which comprises 90% of the protein content of mineralized tissues. Additional matrix proteins in bone include osteonectin, matrix GLA protein, osteocalcin, proteoglycans (decorin, biglycan), bone acidic glycoprotein 75 (BAG 75), osteopontin (2ar, Sppl, pp69), bone sialoprotein (BSP), fibronectin, vitronectin, and thrombospondin (11, 12). The last five proteins contain RGD sequences and are known to mediate cell attachment through interactions with hetereodimeric glycoproteins, termed integrins, that are being characterized on osteoclasts and giant cell tumors (9, 10, 13, 14). Antibody localization suggests that osteopontin mediates Osteoclast attachment to bone through the vitronectin receptor (15); however, the vitronectin receptor also binds bone sialoprotein (16, 17), which mediates attachment of other cell types. Also, the aBs vitronectin receptor is known to be a highly promiscuous integrmn capable of binding to a number of matrix proteins including vitronectin, fibrinogen, fibronectin, and von Willebrands factor (18, 19). BAG 75 is a phosphorylated, acidic glycoprotein synthesized by osteoblasts and is restricted to bone and calcifying cartilage (20, 21). Structurally, BAG 75 appears to be related to osteopontin and bone sialoprotein; however, these phosphoproteins can be distinguished on the basis of calcium binding capacities, i.e., BAG 75 > BSP > osteopontin (Y. Chen and J. P. Gorski, unpublished results). Antibody staining of fetal rat forelimbs and calvaria shows that BAG 75 is enriched in mineralizing areas of new bone (21).

1To whom correspondence should be addressed, Department of Skeletal Diseases, Lilly Corporate anapolis, IN 46285, USA.

at: MC Center,

2Present address: Department of Skeletal Diseases, porate Center, Indianapolis, IN 46285, USA.

620, Indi-

Lilly Cor-

3Present address: 272, Eastern Point 4Present address: try, School of Basic City, Kansas City, 5Abbreviations:

Pfizer Inc., Central Research Division, Box Rd., Groton, CT 06340, USA. Division of Molecular Biology and BiochemisLife Sciences, University of Missouri-Kansas MO 64110, USA. BAG 75, bone acidic glycoprotein; RGD, Argbone sialoprotein; TRAP, tartrate-resistant acid

Gly-Asp; BSP, phosphatase activity; PBS,

phosphate-buffered

MMP-1,

collagenase;

saline;

bPTH,

MMP-3, bovine

stromelysin; parathyroid

hormone.

0892-6638/92/0006-2966/$01 .50.© FASEB

This manuscript represents some of our attempts to understand osteoclast interactions with matrix proteins that regulate bone resorption. Previous experiments with sechistatin - a globular, RGD-containing protein isolated from snake venom - suggested that osteoclast attachment to bone through aBs-like integrin recognition of RGDcontaining matrix proteins was essential for bone resorption (9; M. Sato, W. A. Grasser, P. D. Manno, V. Garsky, J. Murray, and R. J. Gould, unpublished results). However, because the precise role of bone matrix protein (or proteins) that regulates this interaction is unknown, bone resorption assays were conducted to directly test a series of isolated adhesive and phosphoproteins known to be in the osteoclast environment. The data presented here suggest that BAG 75 is an important regulator of bone resorption.

MATERIALS

AND

TABLE

1. Matrix

protein inhibition of chicke,, osteoclastic activity” 1C50, flM

BAG 75 50 BAG Vitronectin

50 100

Thrombospondin Osteopontin

100 100

10

BSP

>100’

Fibronectin

> >

00’

Matrix proteins are ranked according to potency in inhibiting 50% of the resorption activity of isolated chicken osteoclasts, IC20. Student’s #{128} test analysis confirmed the significance of IC,,’s (P < 0.05), except for BSP and fibronectin. ‘Reflects maximum doses examined; actual IC,0’s are higher.

METHODS

spun at 350 x g for 20 mm at 4#{176}C. Cells from below the first band (monocytes) to above the pellet were pooled and resuspended into complete a-MEM with 2.5 ig/ml Chicken osteoclasts were isolated by methods described cytosine-1-B-D-arabinofuranoside (Sigma, St. Louis, Mo.). previously (2, 6). Briefly, medullary bone was harvested from The serum lot was prescreened for maximum resorption acsplit femora and tibiae of laying hens (Dekalb XL) maintivity. Cells were aliquoted at 2 x 106 viable cells/cm2, extained on a calcium-deficient diet (R5070C-9, Purina Mills, cluding erythrocytes, onto culture plates (Costar, CamSt. Louis, Mo.) for 2-4 wk. The cell suspension in bridge, Mass.) or serum-coated no. 1 coverslips (Fisher, phosphate-buffered saline (PBS) (4#{176}C) was passed through a Pittsburgh, Pa.). Bone resorption was quantitated by meas110 tm nylon mesh and then sedimented through a 70% seuring the [3H] release into the media from bone particles rum gradient (Gibco, Grand Island, N.Y.) for 90 mm at 4#{176}C. (20-53 tim) prelabeled in vivo with [3H]proline (2). OsThe bottom 10 ml were cultured in c-MEM (pH teoclasts (1000-5000/cm2) in 48-well plates (Costar) were in7.2) + 10% fetal bovine serum (Gibco) at 37#{176}C for 1 h becubated with 100 g bone/well between days 5 and 6 in the fore extensive washing in PBS. Osteoclasts typically comabsence and presence of test proteins at 37#{176}C. prised 5-30% of the attached cell population. Rat osteoclasts were isolated from the long bones of 1- to For resorption studies, the bottom 5 ml of the serum gra3-day-old neonates, as described previously (22, 23). Briefly, dient was layered over a discontinuous Nycodenz gradient long bones were isolated, split, and scraped into 199 media (Accurate, Westbury, N.Y.) (1.073, 1.099, 1.143 g/cm3) and pH 7.2 with 10% fetal serum (Gibco). After agitation (60 x), the suspension was passed through a 110 tim mesh (Spectrum, Los Angeles, Calif.) and aliquoted onto culture plates (Costar) and serum-coated no. I coverslips (Fisher). Os120’ teoclasts typically comprised more than 1% of these rat cultures. All experiments with rat osteoclasts were conducted within the 24 h after isolation. Rat osteoclastic activity was quantitated by the bone slice resorption assay, as described previously (23). Briefly, bone 0 0. 80 slices (4.4 x 4.4 x 0.2 mm) cut in cross section from steer 0 -0-Osteopontin S femurs were rehydrated with 0.1 ml complete 199 media in BSP 96-well plates (Costar). Osteoclast suspensions were ali-wBAG 75 !eo 50 BAG quoted 0.1 mI/well and incubated for 15 h at 37#{176}C in the ab0 0 Vitronectin -osence and presence of test proteins. Bone slices were then 0 F,b(onectin -.40 devitalized, fixed, dehydrated, and stained with 1% toluidine Thrombospondn -.blue in 1% sodium borate for 1 mm. Resorption lacunae were quantitated by reflected polarized light microscopy (23). Osteoclast

primary

cultures

20

Attachment to

to

Des.

Figure teoclasts (20-53

io

tr6

(M)

1. Matrix protein effects on bone resorption. Chicken os(1-4 x 103/cm2) in the presence of [3H] bone particles tim) were incubated with increasing concentrations of os-

teopontin, bone sialoprotein (BSP), of BAG 75 (50 BAG), vitronectin, din. Resorption (mean ± SEM, n = of [3H] into the media and is

BAG 75, the 50-kDa

fragment

thrombosponas the release plotted as % control with 100% = 24-35 g for resorption measured between days 4 and 6 in culture. For consistency, primary cultures from chickens of the same brood were used to compare these matrix proteins.

BAG 75 INHIBITS RESORPTION

fibronectin, and 3-6) was defined

assay

The attachment of bone to chicken osteoclasts in 48-well plates (Costar) was quantitated as a function of time after addition of 100 pg/well bone particles (20-53 tim) prelabeled with [3Hjproline. Attachment was monitored ± test protein in complete a-MEM media at room temperature. Unbound bone was defined as particles liberated by three washes of cultures with media. The bound particle pool was determined after 4 h incubation in 1 N NaOH, which completely disrupted all cells. Samples were then combusted in a Packard 306 Oxidizer (Packard Instruments, Sterling, Va.) and the [3H] counts were measured with an LKB scintillation counter.

2967

Protein

purification

and

iodination

of BAG

50

75

Bone acidic glycoprotein 75 BSP, and osteopontin were purified from a guanidine HC1-EIYFA extract of young (< 6 months) rat calvariae by methods described previously (21); three separate preparations of each were used with identical results. Purity was assessed by SDS-PAGE before and after radioiodination and by cross-reactivity with specific antibodies. Protein concentrations were determined by amino acid analysis. BAG 75 (3-5 sg) was radioiodinated with 1 mCi of ‘251-labeled Bolton Hunter reagent and dialyzed against PBS before use (24). Monoclonal antibody to BAG 75 (HTP-IVffl1) was radiomodinated by use of chloramine T. Human vitronectin was from Calbiochem (San Diego, Calif.). Human fibronectin was from Collaborative Research, Inc. (Lexington, Mass.). Human thrombospondin was the generous gift of Dr. D. D. Roberts (NIH, Bethesda, Md.).

Control

Osteopontin 40

BAG 75

0)

0

30

0 0

20

0

10

0 [12511BAG

75 binding

assay

with

chicken

0

primary

20

40

60

80

100

120

cultures

Chicken osteoclasts in 48-well plates (Costar) were incubated with 100,000 cpm/well [‘251}BAG 75 in complete a-MEM media containing 0.1% sodium azide and 0-200 nM unlabeled BAG 75. Similarly, virgin 48-well plates (Costar) were incubated with [1251]BAG 75 to determine background levels. Under similar conditions, it had previously been possible to demonstrate specific high-affinity binding of [125I Nle8’18, Tyr3t]bPTH(l-34)NH2 (1400 mCi/timol) to UMR-106 osteosarcoma cells and long bone primary cultures (R. McKee, Merck). Media were removed after incubation and quantitated as the free pool with a Packard 5650 gamma counter (Downers Grove, Ill.). After washing three times with PBS at 4#{176}C, the bound p001 was recovered by treating wells with 0.1M NaOH and counting the solubiized radioactivity.

160

BAG75 D

c 140

Osteopon.

0

120 100 80

o

60

C.)

Time

(mm)

Figure

3. Effects of BAG 75 and osteopontin on the binding of bone particles to chicken osteoclasts. Confluent cultures of chicken osteoclasts were incubated with [3H] bone particles as a function of time in the presence or absence of BAG 75 or osteopontin. Data are presented as sg of bound bone (mean ± SEM, n = 4).

Binding

of BAG

75 to bone

particles

Lyophiized BAG 75 was hydrated (49 g/ml) in a-MEM (Gibco) and then consecutively diluted into siliconized microfuge tubes containing an equal volume of media alone. Rat bone particles (20-53 tim, 100 jig/tube) were immediately added to each tube and the mixtures were incubated at 37#{176}C for 4 h with agitation. Media alone were substituted for BAG 75 in control tubes; all assays were carried out in triplicate. At the end of the incubation period, unbound BAG 75 was removed by two washing steps with 1 ml of warm media containing 0.05% Tween 20; bound protein was pelleted with bone at 500 x g for 4 mm. Monoclonal antibody (10 ji/nil) in media containing ‘251-labeled tracer and 1 mg/ml ovalbumin was then incubated with washed particles for 1 h prior to two washing steps, as indicated above. Bound [‘251]-labeled monoclonal antibody was quantitated by gamma counting before incubation of particles for 1 h with (1/500) horseradish peroxidase goat anti-mouse IgG second antibody (21). After washing steps, assay tubes were developed colorimetrically with substrate (21) for 4 h at room temperature and the A5 was examined by ELISA.

Q20 Autoradiography

0 io-12ihhir10iir9

i-8 Dose

1l;-

i-6

(M)

Figure 2. Effects of BAG 75 and osteopontin on rat osteoclastic activity. BAG 75 and osteopontin were examined by the bone slice assay with isolated rat osteoclasts. Resorption is presented (mean ± SEM, n = 4-8) as % control (absence of added protein) with

100%

=

23-289

lacunae/slice

for rats

from

four

separate

lit-

ters. For consistency, osteodasts from the same litter were used to compare BAG 75 and osteopontin.

2968

Vol. 6

August

1992

Primary osteoclast cultures from rat or chicken long bones on Labtek slides were incubated with 300,000 cpm/well of Bolton-Hunter radioiodinated BAG 75 for 1 h at room temperature in complete 199 media (rat) or a-MEM (chicken) with 0.1% sodium azide. Parallel cultures included further addition of 100 nM unlabeled BAG 75 as planned negative controls. Alternatively, primary cultures also were incubated with 50,000 cpm/well of biologically functional (adenylate cyclase assay with UMR 106 osteosarcoma cells) [‘251-Tyr3t, Nle8’18] bovine parathyroid hormone (1-34) NH2 ± I unlabeled bPTH (1-34), a positive control. High-affinity

The FASEB Journal

SATO E AL.

Figure

4. Substrate binding of [‘251]BAG 75 in the presence of Osteoclasts. Subconfluent primary cultures of chicken osteoclasts (1-4 x 103/cm2) and tissue fragments were incubated with 50,000 cpm/cm2 of [‘251]BAG 75 and increasing concentrations of unlabeled BAG 75 from 0-200 nM. Counts (cpm) adherent to the substrate are plotted minus background ([‘251]BAG 75 absorbed to virgin plates). Rather than a competition curve, a linear increase in the bound pool was observed. Data were fit to a line (r2 = 0.93) by least squares to yield y = 6.16 x - 65.9 with x intercept = 10.7 nM.

1500

I U In

P-

1000

C, 4

C

500

0 perature for 10 acid phosphatase

0 0

100 Total

200 BAG

75

300

(nM)

labeling of osteoblasts (or precursors), which are also found in these primary cultures, was previously shown for PTH (25). After washing three times with PBS at 4#{176}C, cultures were

fixed

in 4%

formaldehyde

in PBS

pH

5.8 at room

tem-

mm

and activity

then

stained

for

tartrate-resistant

(TRAP, Sigma). Slides were washed in H2O and dipped in Kodak NTB2 emulsion diluted 1:1 with 0.6 M ammonium acetate, vertically dried for 2 h at room temperature, and then stored with dessicant at 4#{176}C for 2 wk. The emulsion was developed in Kodak D19 diluted 1:1 with H20 for 4 mm at 15#{176}C, fixed for 4 mm, and rinsed in H2O. Cells and silver grains were imaged by brightfield, differential interference contrast and reflected polarized light microscopy (23, 26).

Figure

5. [1251]BAG 75 autoradiography of osteoclasts. Rat (A, B) and chicken osteoclasts were incubated with 3 x 10 cpm/well [1251}BAG 75 in the absence (A) and presence (B) of unlabeled 100 nM BAG 75. TRAP, multinucleated osteoclasts under either condition showed no significant accumulation of silver granules, as examined by reflected polarized light microscopy (24). For comparison, osteoclasts with three nuclei are shown in A and B. By contrast, some (but not all) clumps of tissue fragments deposited on the substrate appeared to accumulate [‘251}BAG 75 (C). The corresponding micrograph (D) shows the material at the plane of the substrate by differential interference contrast. Bar: 10 tim.

BAG 75 INHIBITS

RESORPTION

2969

Figure 6. BAG 75 staining of rat osteoclasts relative to s-echistatin staining. A) Rat osteoclasts were incubated with 100 nM BAG 75 and stained either with polyclonal or monoclonal antibodies to BAG 75. No significant difference in the fluorescence intensity of experimental was found compared with controls incubated only with second antibody for osteoclasts with three nuclei. ‘B) Rat osteoclasts were coincubated with 0.3 nM s-echistatin and 100 nM BAG 75 and then stained for s-echistatin. No significant difference in fluorescence intensity was observed between these competition specimens and osteoclasts with three nuclei treated only with s-echstatin. Bar: 10 tim.

Fluorescence Rat

microscopy

osteoclasts

on

glass

coverslips

were

incubated

with

100

BAG 75 for 30 mm at 37#{176}C or 0.3 nM s-echistatin for 5 mm at 37#{176}C. Parallel cultures were incubated in 0.3 nM sechistatin for 5 mm at 37#{176}C, followed by washing and incubation in 100 nM BAG 75 for 30 mm at 37#{176}C. Alternatively, 0.3 nM s-echistatin and 100 nM BAG 75 were coincubated for 10 mm at 37#{176}C; longer incubations were not practical because osteoclasts began to detach in s-echistatin after 1 h. Previous experiments (9) showed no evidence of internalization of s-echistatin. Cultures were fixed in 10% formaldehyde, 60 mM PIPES, 25 mM HEPES pH 7.0, 10 mM EGTA, 2 mM MgSO4 for 2 mm, and rinsed in PBS. Cultures were inverted over 50 jil droplets either of polyclonal antibodies (504, 505) to BAG 75, polyclonal antibodies to s-echistatin, or monoclonal antibodies to BAG 75, followed by incubation with fluorescein-labeled goat anti-rabbit and/or rhodaminelabeled goat anti-mouse antibodies. Micrographs were taken with Kodak TMAX-400 (ASA 800) film on a Nikon nM

Microphot

with

Electron

a 60x

planapo

objective

(NA

1.4).

of BAG 75

microscopy

with

an

atomizer

(Effa

spray

mounter,

Fullam,

Schenectady, N.Y.), dried at 1.3 x l0 torr, and rotary shadowed at 6#{176} with platinum-carbon (Denton Vacuum, Cherry Hill, NJ.), as described by Fowler and Aebi (27). Additional samples were processed by C. Franzini-Armstrong (University of Pennsylvania, Philadelphia, Pa.) by layering on

carbon-coated

in cryoprotectant 2970

Vol. 6

glass

coverslips

(30% August

methanol),

1992

graphs

of replicas

or on

cleaved

quick-frozen

mica,

rinsed

in liquid

were

taken

on

a Philips

CM12

at 80

kV.

RESULTS The

functional

matrix

proteins

significance upon osteoclastic

of

purified activity

noncollagenous was evaluated

by

comparing their bone resorption. with [3H]-labeled

actions directly in two in vitro assays of Isolated chicken osteoclasts were cultured bone particles (20-53 tim) in the presence

of

concentrations

increasing

of

osteopontin,

bone

sialoprotein, vitronectin, fibronectin, thrombospondin, and bone acidic glycoprotein 75. Osteoclastic activity in resorption was measured as described by Blair et al. (2). TABLE

BAG 75, lyophilized from 5 mM ammonium formate pH 8.5, was reconstituted to 50 jig/ml in H20 or 0.IM ammonium formate pH 7. After sonication on ice, samples immediately or 6 h after incubation were layered on Formvar coated grids and negatively stained with 2% aurothioglucose, 2% uranyl acetate, or 1% phosphotungistic acid. Alternatively, samples at time 0 and 6 h were sprayed onto cleaved mica

nitrogen, evacuated, and then unidirectionally shadowed with platinum-carbon at 7#{176} in a Balzers (Hudson, N.H.). Replicas were floated off mica with Chlorox (about 1 h) and off glass with HF (47-52%) until the glass dissolved. Grids were washed twice with H2O and then picked up in Photo-fib (Kodak, Rochester, N.Y.) on 400 mesh grids. Electron micro-

2. Pretreatment

effects on resorption’ Resorption,

g

bone

Control

47.3

Pre-clasts Pre-bones Co-add

52 ± 5 28.7 ± 2.6’ 6 ± 0.5’

‘Four parallel sets of cultures of chicken osteoclasts are compared for the effects of BAG 75 on resorption (mean ± SEM, n = 5-6). Pre-clasts refer to cultures pretreated with 50 nM BAG 75 at 37#{176}C for 1 h before washing and incubation with 1#{176}H]-Iabeledbone particles (20-53 tim diameter, 100 pg/well). Pre-bones refer to [31-1]-Iabeled bone aliquots pretreated with 50 nM BAG 75 at 37#{176}C for I h before washing and incubation with osteoclasts. Co-add refers to cultures in which BAG 75 and

I3H]-labeled cultures. controls

The FASEB Journal

bone ‘These as evaluated

particles were added together to osteoclast values were significantly different (P < 0.002) from by Student’s I test analysis.

SATO ET AL.

Specifically, concentrations

[3H]-labeled of matrix

resorption,

and different to adherent cultures of osteoclasts. After 24 h, the amount of [3H] released into the media was assayed as a measure of bone resorption. Controls were treated identically but received no matrix proteins. As shown in Fig. 1, no significant enhancement of resorption was observed; instead, matrix proteins showed an eventual concentration-dependent inhibition of with

little

An exception bone resorption concentrations

was

bone particles in vivo proteins were added

effect

BAG

below

0.1

75, which

jiM.

significantly

inhibited

hibitory

in a concentration-dependent manner at greater than 10 nM. Proteins could be on the basis of their significant (P < 0.05) 50% inconcentrations (IC50’s). As shown in Table 1, BAG

75 and

its 50-kDa

ranked

tent

inhibitors 50 nM.

NH2-terminal

of bone

fragment

resorption,

with

were

the

respective

po-

most

1C50’s

also osteoclasts

observed upon immunofluorescent staining of rat preincubated with 100 nM BAG 75 and followed with anti-BAG 75 monoclonal or polyclonal antibodies. Similar sets of micrographs were obtained of chicken osteoclasts incubated with [125IIBAG 75 (data not shown). Staining of rat osteoclasts (Fig. 6A) was indistinguishable from controls (second antibody alone); immunofluorescence was limited to occasional tissue fragments on the substrate (data not shown). Because osteoclasts are known to interact with RGD-containing proteins (9), competition experiments were conducted with the RGD-containing protein, sechistatin. As shown in Fig. 6B, BAG 75 (100 nM, 37#{176}C, 1 h) was without effect on the s-echistatin-dependent immunofluorescence of rat osteoclasts with three nuclei. Because rat osteoclasts with three nuclei in 100 nM BAG 75 ocwas

of 10

and A similar order of inhibitory potency was obtained with isolated rat osteoclasts, where bone resorption was quantitated by the bone slice assay (23). In order to more closely mimic the in vivo physical environment, rat osteoclasts were aliquoted onto bone slices in the absence or presence of bone matrix

proteins.

Howships

lacunae

h incubation.

BAG

75 displayed

in

with

IC50

this

assay

=

were

a 10-fold 1 nM,

as

quantitated

after

increase

in potency 2; osand rat as-

shown

in

association controls,

(Fig. nM

3). BAG

-fr---

2

75

chicken

4

80000

15

60000 *

40000Osof

*

In c”

20000-

also

had

no

effect

IC50 on

bone

concentration binding

0’

on glass

or plastic

as monitored

until

for

was

observed;

instead,

RESORPTION

(M)

120

1.5

B HTPAb -fr---

E

2 h (data

C

Control

1.0

In 0

0 0

0.5

a concentration-dependent

increase in the substrate bound pool was observed, as shown in Fig. 4. The binding data fit to a line (least squares) revealed an x intercept (critical concentration) of less than 50 nM, above which a linear increase in the substrate bound pool was observed (r2 = 0.943). As values plotted in Fig. 4 were corrected for adsorption of [1251JBAG 75 to plastic, cells or tissue fragments appeared to facilitate the observed linear increase. Autoradiography of rat osteoclasts incubated with 50,000 cpm/well of [1251]BAG 75 alone (Fig. 5A) or with 100 nM unlabeled BAG 75 (Fig. 5B) showed only background labeling of TRAP positive osteoclasts. Levels of silver granules greater than background levels were observed over only some, not all, tissue fragments imaged on the substrate (Fig. 5C-D). A lack of specific binding of BAG 75 to osteoclasts

BAG 75 INHIBITS

-

Concentration

not shown). These data suggest that BAG 75 does not interfere with resorption by substantially inhibiting the kinetics of osteoclast attachment to bone. Binding experiments were conducted with biologically active [1251]BAG 75 to further explore potential interactions with chicken osteoclasts. Subconfiuent primary cultures of chicken osteoclasts (1-4 x 103/cm2 and tissue fragments were used in an effort to approximate physiological conditions. However, no specific competitive binding of [‘25IIBAG 75 to osteoclasts

-

of 10

concentrations (especially of osteopontin) were not used because RGD-containing proteins had previously been shown to affect the spread area of osteoclasts (9), which could artifactually affect the binding of bone to cells. BAG 75 (10 mM) had no effect on the morphology of chicken osspread

*

*

10-10

mm. Higher

teoclasts

*

V..

to isolated chicken had no substantial

resorption

HTP

00000

C

osteoclasts. effect on the kinetics of bone to osteoclasts compared with although partial inhibition was observed at 80 mm

The

A

Control

S.

Fig.

teopontin was similarly effective in the chicken says, with IC50 = 0.1 and 0.6 jiM, respectively. To elucidate the inhibitory mechanism, BAG 75 and teopontin were examined for their effects on the binding bone particles (20-53 tim) Overall, 10 nM osteopontin

120000

10.10

1o9

ii

Concentration

i7

io6

(M)

Figure 7. Direct binding of BAG 75 to rat bone particles. Binding of BAG 75 to rat bone particles (20-53 jim) was monitored by measuring the association of (1251] labeled monoclonal antibody HTP IV#1 with the bone pellet (A). Filled symbols refer to particles treated with BAG 75; open symbols refer to particles treated with media alone. Asterisks above data points indicate a significant difference (P < 0.05, independent Student’s t test) compared to an average of control values. (B) BAG 75 binding to bone particles was estimated in samples (shown in A) with an ELISA assay to measure

total bound size

monoclonal

antibody.

Error bars were smaller

than the

of the symbols.

2971

Figure 8. BAG 75 Forms filamentous structures with time. BAG 75 was sonicated in 0.3 electron microscopy at 0 h (A) and after 6 h (B) incubation at room temperature. Bar: 100 nm.

2972

Vol. 6

August

1992

The FASEB Journal

M

ammonium

formate

(pH 7.0) and processed

for

SATO ET AL.

binding curve is multiphasic, more than 1-10 nM, whereas

30

6 hr

,

>20 U C

0 C. 0

l10

lar

species

between

of 16.8 nm background cies

tion

0 0

20

40

60

80

Size

1 00

1 20

1 40

(nm)

9. Particle size distribution of BAG 75. The particle width of BAG 75 species was measured at 0 h (0-20 mm, n = 55) and 6 h (n = 37), and then plotted with respect to frequency as quantifrom

electron micrographs of low-angle shadowed specimens. Lengths for the 6 h species were not measured because of extreme variability (see Fig. 7B and Fig. 9A-C). Two different distributions were observed with mode = 16.8 nm for a range of 11.3-131.8 nm (0-20 mm) and mode = 26.6 nm for a range of 22.7-53 nm (6 h).

cupied 1.2 ± 0.1 times the spread the spread area of Fig. 5B, Fig. cluded

that

BAG

75 had

area of rat osteoclasts detached rat osteoclasts dence

was

obtained

no

area

of controls

6B with

substantial

Fig. effect

on

the

we conspread

4 nM s-echistatin Therefore, no evihigh-affinity receptors for

specific,

plateau

of

grains

revealed with

11.3 and

8A).

(Fig.

131.8

Smaller

were

not

a positively

in width

skewed

The

(skewness

± 4.1 nm

with

a mode

on the

measured.

of 20.8

a median

nm

structures

order

of

measured 2.113)

=

(Fig.

spedistribu-

9).

and Fig. bA, B). Quantitation of filament diameters revealed a positively skewed (skewness = 1.104) distribution, with mode = 26.6 nm and median = 26.7 ± 8.8 nm for the range of 22.7-53.3 nm diameter (Fig. 9). Striations with a periodicity

of

about

13

nm

were

observed

for

low-angle

shadowed specimens, particularly for the longer filaments (Fig. bOA). Other regions of grids sampled at 6 h showed branched, interconnected structures, suggesting that these filaments could in turn organize into a lattice network (Fig. lOB). In summary, these micrographs suggest that BAG 75 can

self-associate

play

structural

to and

form

filamentous

functional

structures

roles

in

that

the

bone

may

matrix.

(compare

5A),

on glass, whereas within 1 h (9). for

apparent

However, when sampled 6 h after the sonication step, solutions of BAG 75 contained highly ordered filaments, which ranged in length from 23 to more than 1000 nm (see Fig. 8B

Figure

tated

an

became cooperative at higher concentrations. Similar findings are shown in a plot of BAG 75 immunoreactivity (ELISA) on bone partides (Fig. 7B). To examine the possibility that BAG 75 was selfassociating, samples (50-300 nM) were examined by electron microscopy as a function of time by negative stain (aurothioglucose, uranyl acetate, phosphotungstic acid), lowangle (platinum-carbon), and rotary shadowing techniques. Immediately after an initial sonication (or shearing) step (time = 0 h), BAG 75 grids contained heterogeneous globu-

#{149}0 hr

o

with

the binding

soluble BAG 75 on rat or chicken osteoclasts. Nor was evidence obtained for BAG 75 modulation of the aB3-like integrin on osteoclasts (M. Sato et a!., unpublished results). To ascertain whether binding of BAG 75 to bone particles was necessary for the inhibition of resorption, transient incubation experiments were conducted as detailed in Table 2. It is noteworthy that chicken osteoclasts pretreated with 50 nM BAG 75 (1 h, 37#{176}C), followed by extensive washing,

DISCUSSION Bone resorption is recognized to be a multi-step process requiring directed migration of osteoclast precursors to bone, differentiation and recognition of bone sites to be resorbed, attachment and formation of the sealing (clear) zone, cytoplasmic

polarization

with

vectorial

transport

of

lyso-

somes, release of acid and lysosomal enzymes into the apical “ruffled” border compartment, and dissolution of the calcium hydroxyapatite and organic matrix (1-5, 11). In these studies, we attempted to elucidate important organic matrix interactions with osteoclasts by incubating these cells with various matrix proteins known to be in the endosteal bone environresorbed as much bone as controls. However, (3H]-labeled of six purified matrix proteins to bone particles pretreated with 50 nM BAG 75 (1 h, 37#{176}C), ment. The direct addition rat or chicken osteoclast cultures resulted in concentrationand then washed, resorbed significantly less (P < 0.002) dependent inhibition of resorption, whether scored as than controls (55%). These data suggest that a surface BAG resorption lacunae number or as release of [3H] from bone 75/bone complex rather than interactions between soluble BAG 75 and osteoclasts inhibits resorption. particles. RGD-containing proteins were examined because Based on the data depicted in Table 2 and Fig. 4, we of previous suggestions (9, 10, 28) that interactions with hypothesized

that

teoclasts, but that hibits antiresorptive

studies

were

to compare tivity with monitored

BAG

75

a surface activity.

does

not

out with BAG 75 and concentration dependence for direct binding. BAG

carried the that by

bind

radioactivity

antibody HTP IV#1 only substrate-bound

directly

to

os-

complex of BAG 75/bone exTo test this hypothesis, binding

with

rat bone particles of inhibitory ac75 binding was

[‘251]-labeled

resorption

BAG 7S INHIBITS

observed

RESORPTION

in

Fig.

1. The

shape

of

the

proteins

are

critical

for

bone

resorption.

The effects of vitronectin, thrombospondin, and osteopontin on the resorption activity of chicken osteoclasts support this hypothesis because they were equipotent with RGDcontaining s-echistatin IC50 = 100 nM. The in vitro

monoclonal

or by ELISA. This antibody recognizes forms of BAG 75 (21; J. P. Gorski, un-

published results). Significant binding of radiolabeled HTP IV#1 became evident at concentrations above 1 nM (Fig. 7A), which is consistent with the inhibition of chicken osteoclastic

RGD-containing

were

200 nM.

BSP

However,

tion was BAG chicken resorption, ment amino function

75,

least and

(9) in inhibiting resorption with effective in influencing resorption

fibronectin,

of BAG 75 was less and carboxyl-terminal in modulation

with

IC50 of more

than

the most potent inhibitor of bone resorpwith IC50 = 1 and 10 nM for rat and respectively. Because the 50-kDa frageffective portions

of osteoclastic

(IC50 = 50 nM), both of BAG 75 appear to activity.

In no instance

2973

10. BAG 75 forms

Figure

higher

order

structures.

After

6 h, electron

microscopy

samples

of BAG

1 jim long as shown by low-angle shadowing (A). Some areas of grids also showed branching suggesting that BAG 75 filaments can organize to form a lattice network. Some irregular forms that have collapsed onto the gird surface during processing. Bar: 100 nm.

was

enhanced

ticipated sess cell

resorption

observed,

for osteopontin and bone binding and hydroxyapatite

which

may

sialoprotein, binding

have

been

which

an-

pos-

domains.

Osteopontin was suggested by Reinholt et al. (15) to be the matrix protein linking osteoclasts to bone because it was immunolocalized beneath the clear zone. However, some integrins such as the aBs are highly promiscuous in binding to s-echistatin, vitronectin, fibrmnogen, fibronectin, osteopontin, bone sialoprotein, von Willebrand factor, and throm-

2974

Vol. 6

August 1992

bospondin suggests

(9, that

18,

19; M.

osteopontin

75 revealed

some

filaments

more

and interconnections between seen in B may be 3-dimensional

Sato, may

unpublished not

be the

results), only

than

filaments, structures

matrix

which protein

mediating attachment of osteoclasts to bone. is supported by the finding that osteopontin, thrombospondin were nearly equipotent in resorption (IC50 about 1 jiM). In addition, suggest that resorption is not synonymous

This hypothesis vitronectin, and inhibiting bone these data also with attachment

to substrate because although osteopontin largely ineffective in vitro resorption assays,

was found it is active

The FASEB Journal

to be in at-

SATO ET AL.

taching other cells to plastic (16, 17) at input concentrations of 10 nM. In these attachment assays of cells to dishes, osteopontin is adsorbed directly to plastic before incubation

citriol, and PTH (4, 35-41). In organ culture, PTH stimulated the degradation of collagen but not mineral release in the presence of resorption inhibitor acetazolamide (42). In

with

addition,

cells.

The

inability

matrix proteins tion may be due ments

or

of

at higher to the forms

bound

osteopontin

and

concentrations

preference

of some

of adhesion

other

RGD

to inhibit

resorp-

integrins

proteins

for

frag-

(29-32).

The mechanism of BAG 75 inhibition does not appear to involve the aBs-like integrin of osteoclasts because BAG 75 (100 nM) failed to compete with s-echistatin (0.3 nM) binding to this receptor. In fact, specific, high-affinity receptors for

soluble

BAG

75

were

not

observed

on

either

rat

chicken osteoclasts. Rather, preincubation gest that it is the BAG 75/bone surface

experiments complex that

lates

resorption

bone

binding of carrier ism

resorption

activity.

Because

assays were carried protein (i.e., 10%

does not

appear

or

sugmodu-

and

bone

out in the presence of an excess serum), the inhibitory mechan-

to result

from

simply

the

mation more

bone

in

the

presence

process after cytoplasmic

21).

HTP As

of BAG

attachment polarization.

IV#1 recognizes only osteoclasts still attach to

75,

a step

is probably

in

being

the

[3H]-labeled

rat

bone

particles

osteoclasts of bound BAG

and

direct

cultures

of osteoclasts

showed

a rise

75 was

increased

nM.

This

bone

in the

rat

above

suggests to all on bone

osteoclasts

of pooi

differwith the

[‘25I]BAG

containing

a critical

is similar 10 nM

binding

methodological comparison

substrate-bound

beyond

behavior

observed

75

tissue

with

fragments

as unlabeled

concentration

BAG

of 10-50

to the cooperative binding BAG 75 and to polymerization

to

curves derived previously for cytoske!etal proteins such as actin (33). Electron micrographs directly confirmed the formation of highly ordered filaments of 27 nm in diameter from shear-sensitive globular species of turn were observed to branch and after sonication. Because BAG 75 is secreted by riched in areas surrounding newly BAG 75 may function in vivo to degrading

have

shown

newly

forming

that

although

bone.

20 nm. The filaments form lattice networks

The

Merck

Several

osteoclasts

lines

of

will adhere

for

and

for

75 in osteoid

before

of osteoblasts mon pathway

BAG 75 INHIBITS

Grant

1. Chambers,

Res. 6,

thank

Dr. Claire

Franzini-Armstrong

& Dohme Research U. P. G.).

Labs,

and

a UMKC

Faculty

a requirement

resorption. and

stromelysin

(MMP-3)

whose regulated secretion for resorption stimulators,

RESORPTION

are

represents including

colproducts a comIL-i, cal-

The

origin

of the

osteoclast.

Bone Mm.

and inorganic components of bone.] Cell Biol. 102, 1164-1172 3. Blair, H. C., Teitelbaum, S. L., Ghiselli, R., and Gluck, S. (1989) Osteoclastic bone resorption by a polarized vacuolar proton pump. Science (Wash., DC) 245, 855-857 4. Sato, M., and Rodan, G. A. (1989) Bone cell shape and function. In Cell Shape: Determinants, Regulation and Regulatory Role (Stein, W. D., and Bronner, F., eds) pp. 329-362, Academic, Orlando, Florida 5. Baron, R. (1990) Anatomy and ultrastructure of bone. In Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, pp. 3-7, American Society for Bone and Mineral Research, Washington, D.C.

6. Zambonin-Zallone,

7.

9.

10.

11.

its proteolytic

Metalloproteases,

T. J. (1989)

1-25

2. Blair, H. C., Kahn, A. J., Crouch, E. C., Jeffrey, J. J., and Teitelbaum, S. L. (1986) Isolated osteoclasts resorb the organic

evidence

to osteoid,

osteoclast can dean inhibitory role

(MMP-l),

gratefully

of Pennsylvania) for assistance and instruction in samples of BAG 75 for electron microscopy. Portions of were supported by National Institute of Health grant Weldon Spring Fund of the University of Missouri;

Sharp

Research

8. osteoblastic cells and encalcifying osteoid (21), prevent osteoclasts from

mineral that the also consistent with

degradation

authors

(University preparing this work AR40923,

in 6 h

to expose the underlying grade, these results are

lagenase

osteoclasts

We hypothesize that the exposure of BAG 75 on bone surfaces inhibits osteoclastic resorption until it is degraded by proteases, possibly including collagenase and/or stromelysin. Consistent with this hypothesis, preliminary data suggest that stromelysin will produce a 50-kDa fragment of BAG 75; a similarly sized fragment is increased two- to threefold in the serum after ovariectomy in rats (21; J. P. Gorski et al., unpublished results).

resorption does not proceed until the unmineralized layer is first removed by proteases (22, 34). Although the results were interpreted in terms of removal of osteoid collagen in order

BAG

where

for-

perhaps

of responding

are capable 75. Although

slices responded to 1 nM BAG 75, ences may preclude direct quantitative chicken osteoclast data. Attempts to demonstrate binding primary

dentin

the

but not in

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The FASEB Journal

Recewed for publication February 5, 1992. Accepted for publwation April 15, 1992

SATO ET AL.