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Association of Specific Proteolytic Processing of BoneSialoprotein and Bone Acidic Glycoprotein-75 withMineralizationwithin Biomineralization Foci*SReceived forpublication,February 15,2007, andin revised form,June 19,2007 Published, JBC Papers in Press,July 5, 2007, DOI 10.1074/jbc.M701332200

Nichole T. Huffman, J. Andrew Keightley, Cui Chaoying, Ronald J.Midura, Dinah Lovitch, PatriciaA. Veno,Sarah L. Dallas, and Jeff P.Gorski1

From theBone Biology Program, Department of Oral Biology, School of Dentistry, University of Missouri,Kansas City, Missouri 64108, Biological Mass Spectrometry and Proteomics Facility, Division of MolecularBiology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110,Tibet University Medical College, Lhasa 850002, Tibet, China, and the Department of Biomedical Engineering,Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195

Mineral crystal nucleation in UMR 106-01 osteoblastic cul-

tures occurs within 1525-m extracellular vesicle-containing

biomineralization foci (BMF) structures. We show here that

BAG-75 and BSP, biomarkers for these foci, are specifically

enriched in laser capture microscope-isolated mineralized BMF

as compared with the total cell layer. Unexpectedly, fragments

of each protein (4550 kDa in apparent size) were also enriched

within captured BMF. When a series of inhibitors against differ-

ent protease classes were screened, serine protease inhibitor

4-(2-aminoethyl)benzenesulfonylfluoride HCl (AEBSF) was the

only one that completely blocked mineral nucleation within

BMF in UMR cultures. AEBSF appeared to act on an osteoblast-

derived protease at a late differentiation stage in this culture

model just prior to mineral deposition. Similarly, mineraliza-

tion of bone nodules in primary mouse calvarial osteoblastic

cultures was completely blocked by AEBSF. Cleavage of BAG-75

and BSP was also inhibited at the minimum dosage of AEBSFsufficient to completely block mineralization of BMF. Two-di-

mensional SDS-PAGE comparisons of AEBSF-treated and

untreated UMR cultures showed that fragmentation/activation

of a limited number of other mineralization-related proteins

was also blocked. Taken together, our results indicate for the

first time that cleavage of BAG-75 and BSP by an AEBSF-sensi-

tive, osteoblast-derived serine protease is associated with min-

eral crystal nucleation in BMF and suggest that such proteolytic

events are a permissive step for mineralization to proceed.

Bone is a vascularized tissue that uniquely becomes min-

eralized as part of its developmental program (1). Mineral-

ized bone serves essential vertebrate functions, including

structural support, reversible storage for calcium and phos-

phorus, and as a reservoir for toxic metals and carbonate (2).

Bone tissue is composed of osteoid; osteoblasts, which pro-

duce and mineralize new bone; osteoclasts, which resorb

bone; and osteocytes, mature osteoblasts that maintain bone

viability (13). Osteoid is a type I collagen-rich extracellular

matrix enriched in acidic noncollagenous proteins (4). Using

fetal rat calvaria cell cultures, Bellows et al. (5) showed that

osteoid is unmineralized when initially deposited, and min-

eral crystals form within nodular structures over the follow-

ing 4872 h. Bone matrices can be classified as lamellar,

based on a highly organized layered structure, or woven

bone. Woven bone is formed during embryonic develop-

ment, fracture healing, and at sites receiving mechanical

stimulation in excess of 3,000 microstrain (6); lamellar bone

replaces woven bone later in development.

The question of whether bone mineralization is under

direct osteoblastic control or whether it is purely a passive

chemical process is under active investigation. Schinke et al.

(7) have proposed that calcification reactions in vivo are pas-

sive physiochemical processes occurring readily where local

mineralization inhibitors are overwhelmed. In support of

this hypothesis, Murshed et al. (8) produced a calcified der-

mal layer in transgenic mice expressing alkaline phosphatase

in skin under the control of the type I collagen chain pro-

moter (2). Similarly, Luo et al. (9) and Murshed et al. (10)

showed that matrix GLA protein is a passive local inhibitor

of vascular calcification because deficient mice calcify their

thoracic aorta. The latter approach emphasizes the forma-

tion of hydroxyapatite crystals as the primary experimental

outcome.

A second view focuses on the active role of local extracel-

lular nucleation complexes such as biomineralization foci

(11, 12), crystal ghosts (13, 14), matrix vesicles (15), and the

hole regions of collagen fibrils (16) with matrix vesicles (17,

18) or with extracellular matrix phosphoproteins (12, 19,

20). We have proposed that mineralization can be divided

*This work was supported by National Institutes of Health Grants R21 DE14619

(to J.P. G.) and R01 AR052775(to J.P. G.) and small grants from the WomensCouncil of the University of Missouri, Kansas City (to N. T. H.). Parts of thisresearch were presented in preliminary form at the annual meeting of theAmerican Society for Biochemistry and Molecular Biology, June 1216, 2004,Boston, MA;the American Society for Boneand Mineral Research, September2327, 2005, Nashville, TN; and the American Society for Bone and MineralResearch, September1519,2006,Philadelphia,PA. Thecostsof publicationofthis article were defrayed in part by thepaymentof page charges. This articlemust therefore be hereby marked advertisement in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

S The on-line version of this article (available at http://www.jbc.org)containssupplemental Figs. S1S3.

1To whom correspondence should be addressed: Bone Biology Program,Oral Biology, School of Dentistry, 625 East 25th St., University of Missouri,Kansas City, MO 64108. Tel.: 816-235-2537; Fax: 816-235-5524; E-mail:[email protected].

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 36, pp. 2 600226013, September 7, 2007 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

26002 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 NUMBER 36 SEPTEMBER 7, 2007

http://www.jbc.org/content/suppl/2007/07/06/M701332200.DC1.htmlSupplemental Material can be found at:


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into a cell-mediated nucleation phase within BMF,2 followedby passive growth and expansion of these initial crystals (11,

12). In this model, once the initial crystals reach sufficientsize and number, the BMF barrier function is abrogated,facilitating the passive growth and expansion of the initialmineral phase into the larger, territorial collagenous matrix.The latter research focuses on the in vivo functionality of the

mineralized bone product (1019). In this context, hydroxy-apatite crystal formation is envisioned to occur in a mannerthat facilitates subsequent vascular access to the crystals andplacement of crystals within the organic matrix so as to facil-itate mechanical support for organs, joints, muscles, and

tendons.Bone osteoid is enriched in phosphoproteins, acidic glyco-

proteins, and proteoglycans, some of which like BSP or its frag-ments are nucleators of hydroxyapatite crystals (20, 21). Wehave shown that phosphoglycoprotein BAG-75 expression

delineates future extracellular sites of mineralization in vivowithin woven bone and in vitro termed BMF (11, 12). BMF are1525-m spherical extracellular structures containing several

sizes of vesicles (15), which are sites of the first mineral crystalsin the UMR osteoblastic model (12). Following plating, UMRcells proliferate and differentiate over the first 6064 h andattain a competency to initiate mineralization in BMF, if sup-plemented with a phosphate source (22). BSP has also beenlocalized to mineralizing nodules termed crystal ghost aggre-

gates in rat bone, which are analogous to BMF (13, 14, 23). BSPis incorporated into BMF just prior the appearance of mineralcrystals (12, 22). Based on these findings, we proposed thatBMF structures function in an active mineralization processinitiated and controlled by osteoblastic cells.

To better understand the role of BMF in bone mineral nucle-ation, we have begun to characterize the proteome of mineral-ized BMF isolated by laser capture microscopy. Our resultsshow that isolated BMF are not only physically enriched in

BAG-75 and BSP but also fragments of each. Screening inhibi-tors of the different classes of proteases revealed for the firsttime that serine protease inhibitor AEBSF completely blockedcleavage of BAG-75 and BSP, as well as mineral crystal nucle-ation within BMF. Two-dimensional SDS-PAGE comparisons

of AEBSF-treated and control cultures suggested that activa-tion of procollagen processing may also be inhibited. Takentogether, our results demonstrate an association between ser-ine protease cleavage of mineral nucleator BSP and mineralcrystal nucleation within biomineralization foci and mineral-

ization nodules.

EXPERIMENTAL PROCEDURES

Materials

Antibodies were from several sources as follows: nonim-mune rabbit IgG (EMD Biosciences), anti-BAG-75 (number

503) (anti-peptide antibody) rabbit serum (24); anti-BAG-75

(number 504) (anti-protein antibody) rabbit serum (24); anti-bone sialoprotein LF-100 antiserum (Larry Fisher, NIDCR,

National Institutes of Health); and monoclonal anti-BSP(WV1D1(9C5)) antibody (NIH Developmental Studies Hybri-

doma Bank, University of Iowa).

Methods

Cell CultureUMR 106-01 BSP cells were passaged and cul-tured at 37 C and 5% carbon dioxide as described previously

(12, 22) and updated briefly here. Cells were seeded at a densityof 1.0 105 cells/cm2 in Growth Medium (Eagles MEM sup-

plemented with Earles salts, 1% nonessential amino acids(Sigma), 10 mM HEPES (pH 7.2), and 10% fetal bovine serum(Hyclone)). After 24 h, the medium was exchanged with

Growth Medium containing 0.5% BSA (catalog numberA-1933, Sigma) instead of FBS. Sixty four hours after plating,

the culture medium was exchanged with Mineralization Media(Growth Medium containing either 0.1% BSA or 10% fetal

bovine serumand 7 mM BGP). Cultures were then incubatedforan additional 24 h, at the end of which (88 h) the cells were

either subjected to MTT assay or fixed in 70% ethanol and thenextracted for protein. In some experiments, protease inhibitors,

including serine protease inhibitor AEBSF [(4-(2-aminoethyl)-benzenesulfonylfluoride HCl)] (EMD Biosciences), were added

to cultures at 64 h after plating in Mineralization Media. Alter-natively, AEBSF was added at 44 h after plating; inhibitor was

then removed and exchanged for Mineralization Media at 64 hand the amount of mineralization analyzed at 88 h.

Primary mouse osteoblasts were isolated from calvaria of57-day-old mice using a modification of themethod described

previously (25, 26). Briefly, the calvaria were aseptically har-vested, and four sequential 20-min digests were performed in

0.05% trypsin, 0.2% collagenase in Hanks balanced salt solu-tion. Fractions 24 were pooled, centrifuged, and resuspended

in -MEM containing 10% fetal bovine serum, 2 mM L-gluta-

mine, 100 units/ml penicillin, and 30 g/ml gentamicin(-Growth Medium). 2 106 cells were plated per T-75-cm2

flask and allowed to reach confluency (34 days). Confluentflasks were then trypsinized and plated into 12- or 24-well cul-

ture dishes for experiments at a density of 20,000 cells per cm2

growth area using media and supplements as described above.

At confluency, the media were changed to -MEM containing5% FBS, 50 g/ml ascorbic acid, 5 mM BGP, and other supple-

ments as described above. BGP was omitted from some wellsthat served as an un-mineralized control. To test the effect of

AEBSF, identical duplicate cultures were treated on days 3, 6, or9 with 0.003 to 0.1 mM AEBSF. Phase contrast images weretaken of living cultures on days 312. On day 12 after plating,

one set of cultures was incubated with MTT as described belowto determine cell viability. A second set of cultures was fixed on

day 12 with 70% ethanol and processed for quantitativeAlizarinred S staining as described below.

2The abbreviations used are: BMF, biomineralization foci; AEBSF, 4-(2-amin-oethyl)benzenesulfonylfluoride HCl; FBS, fetal bovine serum; BSP, bonesialoprotein; BAG-75, bone acidic glycoprotein-75; BGP, -glycerol phos-phate; BSA, bovine serum albumin; CL, total cell layer extract fromBGP-treated cultures; CL, total cell layer extract from cultures nottreated with BGP; MAA, Maackia amurensis agglutinin; MS/MS, tandemmass spectrometry; LC, liquid chromatography; SKI-1, subtilisin kexinisozyme-1; LCM, laser capture microscope; 1,25D3-MARRS, 1,25-vita-min D3-membrane-associated rapid-response steroid-binding protein;CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonicacid; CAPS, 3-(cyclohexylamino)propanesulfonic acid; MTT, 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MEM, minimalessential medium; MAA, Maackia amurensis agglutinin.

Proteolytic Processing Is Essential forMineralizationof BMF

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MTT AssayCulture wells were washed with Eagles MEMsupplemented with Earles salts and then incubated with a solu-

tion of 0.5 mg/ml MTT in Eagles MEM for 12 h at 37 C (27).Residual MTT solution was removed; the cells were disruptedby mixing briefly with dimethyl sulfoxide, and free reduced dyewas read at 490 or 540 nm in a spectrophotometer.

Quantitation of MineralizationAfter fixation in 70% etha-

nol, the cell layer was rinsed and stained with Alizarin red S dyeas described previously (22). The same procedure was also usedfor serum-depleted cultures with the following modified wash-ing protocol, e.g. the stained cell layer was rinsed once with 1mM HEPES in nanopure water. A standard curve for Alizarinred S dye was constructed for each analysis, and amount ofbound dye/culture well determined.

Statistical MethodsAll statistical tests were performedusing SigmaStat 3.1 software (Systat Software, Inc.). A one-way

analysis of variance test was used to determine whether a sta-tistical difference existed between the viability of UMR-106-01cultures or the amount of mineral deposited. Subsequent pair-wise multiple comparison tests were performed with the Stu-

dent-Newman-Keuls or the Kruskal-Wallis method.Extraction of Cell Layer Fraction; One-step MethodCells

were dislodged by scraping and then extracted with 75 mMpotassium phosphate buffer (pH 7.2), containing 10 mMCHAPS, 75 mM sodium chloride, 50 mM tetrasodium EDTA, 10

mM benzamidine hydrochloride, 2 mM dithiothreitol, and0.02% sodium azide for 1 h at 4 C. Each extract was thenhomogenized briefly using a motorized pestle and clarified byultracentrifugation at 30,000 rpm for 1 h at 4 C in an SW 50.1rotor prior to use. Conditioned media were immediately heated

at 95 C for 5 min to inactivate protease activity and frozen at80 C until analyzed.

Extraction of Cell Layer; Two-step MethodDuring the final24-h mineralization period, cells were grown in BSA-free,serum-free media conditions to reduce the amount of BSA in

fractions used for two-dimensional gel electrophoresis. Mediawere removed from each flask, heated at 95 C for 5 min, dia-lyzed against 5% acetic acid, and lyophilized to dryness. Celllayers were first extracted without mixing for 2 h at 4 C in0.05 M Tris acetate buffer (pH 7.5) containing 0.15 M NaCl,0.05 M EDTA, and 0.02% sodium azide; extracts were theninactivated at 95 C for 5 min, dialyzed against 5% HAc, andlyophilized to dryness. The residual cell layer was next dis-lodged by scraping and extracted overnight at 4 C by slow

mixing with 0.1 M Tris acetate buffer (pH 7.5) containing 8 M

urea, 2% (w/v) CHAPS, and 0.02% sodium azide. Ureaextracts were homogenized and clarified by ultracentrifuga-tion at 30,000 rpm for 1 h at 4 C in an SW 50.1 rotor prior touse in two-dimensional gel electrophoresis.

Western Blotting Chemiluminescence DetectionCell layerextracts and media fractions prepared as described above wereelectrophoresed under reducing conditions on 420% lineargradient gels (ISC BioExpress) according to Laemmli (28) andelectroblotted onto polyvinylidene difluoride membranes (Mil-

lipore Corp.) for 2 h at 100 V.The transfer buffer was composedof 10 mM CAPS buffer (pH 11.0) containing 10% methanol.Blots were processed essentially as described previously (29).Immunoblotting with digoxygenin-MAA lectin followed a pro-

cedure described earlier (29) except that a secondary horserad-ish peroxidase-conjugated anti-digoxygenin antibody was used

for chemiluminescent detection. Films were digitized using aflat bed scanner. To detect directly acidic bone glycoproteins orphosphoproteins, some gels were stained with Stains All (29).

Laser Capture MicroscopyUMR cells were grown as usualon Fisher Plus microscope slides (Fisher), fixed, and stained

with Alizarin red S dye. Immediately prior to laser capture,slides were dehydrated through a graded series of ethanolwashes and xylene rinses. Dried slides were stored at 20 C ina sealed box with desiccant until used. Mineralized BMF werecollected onto standard caps using an Arturus Pixel IIe micro-

scope. Collection films were pooled and stored in 70% ethanolat 20 C until 6200 BMF were collected. LCM-capturedBMF were then mixed in 70% ethanol to dislodge the purple-stained particles that were then microcentrifuged to removethe ethanol. BMF pellets were extracted twice sequentially over

a2-dayperiodat4 Cwith1.1mlof0.1 M Tris acetate buffer (pH7.8) containing with 0.5% octyl glucoside, 0.05% SDS, 0.05 MEDTA, and 0.02% sodium azide. Extracts were then dialyzed

first against 0.01 M Tris acetate buffer (pH 7.8) containing 8 Murea, 0.05% SDS, 0.1% octyl glucoside, 0.05 M EDTA, and sec-ond against 0.01 M Tris acetate buffer (pH 7.8) containing 8 Murea, 0.05% SDS, and 0.1% octyl glucoside. Controls consistedof glass slides containing the total cell layer fractions fromBGP orBGP cultures; control slides were extracted using asimilar protocol. The resultant dialyzed extracts were used forcomparative blotting studies where identical protein amountswere loaded per gel lane.

Protein DeterminationProtein concentration of BMF

extracts was determined using the NonInterfering ProteinAssay by Geno-Technology Inc. (St. Louis, MO).

Mass Spectrometric AnalysesProtein bands and spots weredetected by staining with Coomassie Blue G dye or with SyproRuby dye according to the manufacturers instructions (Bio-

Rad). Excised gel bands/spots were reduced and alkylated fol-lowed by digestion with trypsin for 616 h (30). Peptides wereextracted and subjected to reverse phase capillary liquid chro-matography-mass spectrometry with a linear 270% acetoni-trile gradient over 45 min in 50 mM acetic acid, in a 50m innerdiameter 7 mm Phenomenex C18 Jupiter Proteo capillarycolumn. The column eluted directly into an LTQ linear ion trapmass spectrometer as described previously (30). The instru-ment was operated in the data-dependent mode in which one

mass spectrum and eight collision-induceddissociation spectra

were acquired per cycle. The data were analyzed using Mascotprotein identification software (Matrix Science), with manualinspection of data base matches for validation. The Mascotidentification program (Matrix Science) uses a statistical

method to assess the validity of a match (31). Criteria used forprotein identifications include matching the peptide based onthe following: 1) the precursor (peptide) mass, and 2) MS/MSfragment masses present in the scan, coinciding with thepredicted masses of peptides (and peptide fragment masses)

from a data base entry. Protein searches are currently basedon comparison to all or a subset of (rodent, for example) thesequences present in the MSDB data base, filenameMSDB_20050227.fasta (February 27th, 2005 version). Protein





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identifications made contain at least two peptides match in theMS/MS scans that meets or exceeds the threshold values for a

95% confidence level.Two-dimensional PAGEGels were run according to the

method of Witzmann et al. (32) and stained with either colloi-dal Coomassie Blue G, Pro-Q Emerald 300 glycoprotein stain(Invitrogen), or Pro-Q Diamond phosphoprotein stain (Invitro-

gen). PD-Quest (Bio-Rad) software wasused to digitally analyzethe colloidal Coomassie Blue G-stained gels comparing AEBSF-treated with nontreated cell layer and media fractions to iden-tify proteins differentially expressed in one condition versusanother.

RESULTS

Mineralization of UMR Osteoblastic Cells Is Unchanged in

Serum-depleted ConditionsTo limit contamination by serumproteins in isolated BMF, we tested whether use of serum-freeconditions would affect the amount or morphology of mineral-ization in UMR cultures. No differences were noted in theamount or morphologyof mineralized BMF when conditions of

serum depletion were compared with serum-replete conditions(compare Fig. 1, A versus C). As expected (22), few mineralcrystals are evident when BGP is omitted (Fig. 1B). Quantita-tion of the amount of Alizarin red stain bound per well alsorevealed no significant differences (not shown). Manual counts

of mineralized BMF formed under serum-containing andserum-depleted conditions showed no statistical difference(103 foci/cm2 6.56 S.D. versus 105 mineralized foci/cm2 6.08 S.D.,p 0.486 using one-way analysis of variance followed

by Kruskal-Wallis method). These results confirm that themineralization potential is unchanged in conditions of serumdepletion.

BAG-75 and BSP and Fragments of Each Are Enriched within

Purified Mineralized BMFMineralized BMF, which appear as

dark spots about 2025 m in diameter, were isolated fromethanol-fixed, Alizarin red-stained UMR cultures by laser cap-ture microscopy (Fig. 1, DF). Use of Alizarin red staining foridentification provides a direct connection to previous work

that defined BMF structures as sites of initial mineral crystalnucleation (12, 22).After laser capture of mineralized BMF, Fig.

1Edepicts residual holes that are devoid of cells and show theunderlying glass surface. Finally, Fig. 1Fprovides an image ofthe resultant captured BMF preparation with individual fociarranged in the same relative orientation as they appeared on

the original stained slide (Fig. 1D). Visual inspection of cap-tured populations revealed an absence of obvious cellular con-tamination. Furthermore, attempts at direct isolation of cellsfrom the fixed cell layer using an LCM approach proved unsuc-cessful demonstrating that the fixed UMR cells adhere too

tightly to the glass slide to permit their capture from this

surface.3

Following capture, 6200 pooled BMF were extracted bymixing with 0.1 M Tris acetate buffer (pH 7.8), containing with

0.5% octyl glucoside, 0.05% SDS, 0.05 M EDTA, and 0.02%sodium azide, and then subjected to SDS-PAGE. As controls,UMR culture slides containing the total cell layer along withmineralized BMF (as depicted in Fig. 1D,CL) were processedsimilarly. Culture slides not treated with -glycerol phosphate

and containing nonmineralized pre-BMF represent a secondcontrol (as depicted in Fig. 1B,CL). Equal amounts of proteinwere applied to each lane, and gels were stained as indicated(Fig. 2).

SDS-PAGE results show substantial enrichment of 75-kDaglycoproteins and phosphoproteins in the BMF extract whencompared directly with theCL control (Fig. 2, see bands withasterisks). Sypro Ruby staining showed enrichment of bands inthe 65-kDa range in BMF. In contrast, bands in the 1015-kDarange appearedto be sharedby boththe BMF and total cell layer

samples (Fig. 2). Although not quantitative, this comparativeanalysis is designed to identify those proteins substantiallyenriched within mineralized BMF. Our approach is based uponthe hypothesis that BMF are structures assembled for the spe-cific purpose of nucleating hydroxyapatite crystals in culture

3 N. T. Huffman and J. P. Gorski, unpublished results.

FIGURE 1. Biomineralization foci can be isolated from total cell layersby laser capture microscopy. A and B, UMR-106-01 osteoblastic cellswere cultured in serum-depleted conditions (BSA), or C, the presence ofserum (FBS). Cultures were stained with Alizarin red S to detect hydroxy-apatite crystals. B, bothconditions failed to mineralizein the absenceof BGP.

Arrows point to mineralized BMF (A and C). Scale bar 500 m. DF, lasercapture microscopy of Alizarin red S stained BMF from UMR-106-01 culture.

Arrows refer to the same BMF structures in all panels. D, microscopic view offield to be laser-captured. E, appearance of the residual cell layer left behindafter laser dissection of mineralized BMF. F, purified BMF temporarily affixedto the cap used for laser capture. Gel images are representative of multipleanalyses on two separate BMF preparations. Scale bar 25m.

FIGURE 2. LCM-capturedBMF display distinctive glyco- and phosphopro-tein staining patterns compared with the total cell layer fraction. Thesame amount of protein was applied to each lane. Asterisks emphasize thequantitative enrichment of 7580-kDa glycophosphoproteins and of a65-kDa Sypro ruby-stained protein in BMF lanes. Molecular weight estimatesrefer to blue pre-stained standards co-electrophoresed on the same gel.Results depictedare representativeof two separateBMF preparations. Buffer,0.1 MTris acetate buffer (pH 7.8), containing with 0.5% octyl glucoside, 0.05%SDS, 0.05 M EDTA, and 0.02% sodium azide); BMF, represents proteinsextracted from purified BMF (see Fig. 1F); CL, cell layer extract of culturesafter 24h mineralization in-glycerol phosphate(see Fig. 1D);CL, cell layerextract of cultures not treated with -glycerol phosphate (see Fig. 1B).





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and in primary bone (11, 12). Because mineral nucleation is aspecialized function, our hypothesis predicts that BMF should

exhibit a specialized proteome. Existence of clear differences(more than 510-fold) in 75-kDa glyco- and phosphoproteinsbetween the BMF proteome and that of the CL control sup-ports this hypothesis. The absence of similar post-translation-ally modified proteins in theCL control re-enforces this find-

ing (Fig. 2).Immunoblotting studies (Fig. 3, AE) revealed that the75-kDa glycophosphoproteins BAG-75 and BSP were both dra-matically enriched in BMF only in the presence of BGP. Closerinspection reveals BMF fractions also contain a higher relative

content of BAG-75 and BSP fragments (Fig. 3, B, D, and E,arrows). In the case of BAG-75,this was detectedthrough use ofan N-terminal 3-13 anti-peptide antibody (number 503), whichis known to preferentially recognize a 50-kDa fragment (24).For BSP, a 4550-kDa fragment was observable when the full-

length BSP band was purposely overloaded (Fig. 3, CE). Non-mineralizing cultures also contain a much smaller amount ofthe 4550-kDa fragment (Fig. 3, D and E, CL), although the

gel band pattern is different from that for cultures treated withBGP (CL). These findings validate the use of laser capture

microscopy as a means to purify mineralized BMF from UMR106 cell monolayers. Enrichment of full-length protein withinBMF links BAG-75 and BSP with mineral nucleation, whereaslocalization of their cleavage fragments at the site of initial crys-tal nucleation raises a question as to whether proteolytic cleav-

age of BAG-75 and BSP is required for mineral nucleationwithin BMF.

Results with whole animals indicate that BAG-75 and BSPare two major glycoproteins in rat bone. Specifically, total 4 Mguanidine HCl, 0.5 M EDTA extracts of the mineralized com-partment of bone (33) contain a single 75-kDa glycoproteinband reactive with MAA lectin (Fig. 3F). This result parallelsthat obtained upon glycoprotein staining of UMR fractions

(Fig. 2). Bone extracts, like UMR extracts, also contain a majorphosphoprotein of this size revealed after Stains All staining(Fig. 3F). Finally, as shown in Fig. 3H, both purified BSP andBAG-75, but not a characteristic 50-kDa fragment of BAG-75(24), strongly react with MAA lectin. As a result, we conclude

that BAG-75 and BSP together compose the 75-kDa glycoph-osphoprotein band whose cellular distribution specificallyreflects the state of mineralization in the UMR culture model.

Serine Protease Inhibitor AEBSF Inhibits Mineral Crystal

Nucleation in UMR 106 and in Primary Mouse Calvarial

CulturesTo investigate the nature of the protease activityresponsible for BAG-75/BSP cleavage and the relationship ofcleavage with mineralization, we tested a variety of proteaseinhibitors (Table 1) in the UMR model. Individual inhibitorswere added to confluent cultures at 64 h after plating, and the

amount of mineral deposited within BMF was quantitated 24 hlater. UMR cultures are not competent to mineralize until6064 h after plating, reflecting an osteogenic differentiationprocess that leads to the production of spherical pre-BMFstructures.4

4 J. P. Gorski and R. J. Midura, manuscript in preparation.

FIGURE 3. Comparative immunostaining of LCM-isolated BMF versustotalcell layerfractions demonstratesan enrichment of BAG-75 andBSPand their fragments. AE, protein extracts from LCM-captured BMF, celllayer fractions, and a buffer control were electroblotted onto polyvinylidenedifluoride membrane and developed with anti-BAG-75 or anti-BSP antibod-ies. Arrows indicate 4550-kDa fragments of BAG-75 and of BSP enriched inBMF extracts over that in the CL total cell layer. The amount of proteinloaded onto gel lanes for blots ACwas 6.5 g, for blot D was 13 g, and forblot Ewas26g. BlotsD and Ewereintentionallyoverdevelopedto detect the

BSP fragment. Antibodies used are as follows: anti-BAG-75 protein antibody(number 504); anti-BAG-75 peptide antibody (number 503); and anti-BSPantibody (LF-100). BMF, extract of LCM-captured biomineralization foci;CL,extract of total cell layer from -glycerol phosphate-treated cultures; CL,extract of total cell layer from cultures not treated with -glycerol phosphate;buffer, 0.1 M Tris acetate buffer (pH 7.8), containing with 0.5% octyl glucoside,0.05% SDS, 0.05 M EDTA, and 0.02% sodium azide. FH, BAG-75 andBSP aretwoprominent 75-kDa glycoproteins in rat primary bone extracts. F, rat bone G/Eextract was electroblotted and detected either with digoxygenin-labeled MAAlectin or with anti-BAG-75 antibodies (number 504) (immunoblot). The extractwasalsoelectrophoresedand stained directlywith Stains Alldye. G, purifiedBSP,BAG-75, and mixture of BAG-75 and its 50-kDa fragment were electrophoresedandstainedwithStainsAll. H,purifiedBSPandamixtureofBAG-75andits50-kDafragmentwere electroblotted and detectedwith digoxygenin-labeled MAAlec-tin. Molecular weight estimates are based on pre-stained standards co-electro-phoresed on thesame gel.





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Only one inhibitor, AEBSF, blocked mineral nucleation in

BMF (Table 1 and Fig. 4A). AEBSF is a covalent serine proteaseinhibitor (34) and was capable of completely blocking mineralnucleation at concentrations as low as 0.04 mM. None of theother protease inhibitors tested, which included inhibitors of

thrombin, plasmin, plasminogen activator, furin, and matrixmetalloproteinases, diminished mineralization in the UMR sys-temwhen used at their optimal recommended dosage(Table 1).When added at 64 h after plating, AEBSF was similarly effectiveregardless of whether serum was included in the culture mediaor not (Fig. 4A), indicating that the source of the mineraliza-

tion-related, AEBSF-sensitive protease is the UMR 106 cellsthemselves. However, the time at which AEBSF was added dra-matically influenced the outcome. Assuming a control minerallevel represented by 150170 nmolof Alizarin red dye/well, theinhibitor was 10-fold less effective if present during the period

in which the cells are actively proliferating and differentiating(4464 h after plating) rather than during the mineralizationperiod (64 88 h after plating) (22) (Fig. 4A).

To exclude the possibility that the effects of AEBSF werebecause of cell toxicity, AEBSF-treated and nontreated control

cultures were analyzed using the MTT assay, a widely acceptedassay for cell viability that measures vital mitochondria (27). Asshown in Fig. 4A, AEBSF only shows toxic effects at concentra-tions above 0.4 mM. This outcome is similar whether cells aregrown in serum-sufficient or serum-depleted conditions.

Previously, we have shown that mineralization of MC3T3osteoblastic cells and of primary calvarial osteoblasts alsooccurred at sites enriched in BAG-75 (12), similar to that in the

UMR model. We therefore next determined whether AEBSF

could also block mineralization in primary calvarial osteoblasts.Because the length of exposure is necessarily longer than withUMR cells because of the longer time course of this mineraliza-tion model, we initially added AEBSF starting at day 0, 3, 6, or 9

and continuing until the end of the 12-day culture period (notshown). Media-containing treatments were changed every 3days. On this basis, the minimal effective treatment windowwas found to be between days 9 and 12 (Fig. 4B). Specifically,0.010.1 mM AEBSF was able to block mineralization com-

pletely in primary calvarial cultures when scored on day 12 (Fig.4B and Fig. 5). Parallel assays of primary calvarial osteoblastviability showed that AEBSF had no consistent effect on cellviability (Fig. 4B). Instead, cell viability seemed to exhibit a very

gradual decrease over the concentration range tested and

approached 90% of the control value at 0.1 mM AEBSF. Miner-alization of primary calvarial osteoblast cultures occurs withinmultilayered nodules(Fig. 5),whereas in UMR106 cultures,it isinitiated within spherical 2025-m BMF structures. Despite

the clear morphological differences between these two sites ofmineral nucleation, AEBSF was similarly effective in both sys-tems (Fig. 4, A and B).

Uptake of a 75-kDa Phosphoglycoprotein Band in the Cell

Layers of Mineralizing Cultures Is Blocked by AEBSFToexamine the effect of AEBSF on the protein distribution within

mineralizing UMR 106 cultures, cells were grown until 64 h atwhich time they were re-fed using one of the following fourdifferent media conditions: 1) 7 mM BGP only (BGP);2) 7 mMBGP and 0.1 mM AEBSF (BGP AEBSF); 3) the absence of

both BGP and AEBSF (BGP), and 4)with 0.1 mM AEBSF pres-ent but no BGP (BGP AEBSF). Cell layer extracts andmedia fractions from all four conditions were then comparedusing SDS-PAGE followed by staining or immunoblotting.

Following mineralization, the cell layer was extracted with

eithera50mM EDTA/CHAPS detergent solution or with an 8 Murea, 0.5% SDS, 50 mM EDTA extraction solution (see Exper-imental Procedures for details). Solubility was defined byultracentrifugation at 108,000 gfor 1 h. Gel electrophoresisrevealed that during the 24-h mineralization period (BGP

AEBSF), a 75-kDa glyco- and phosphoprotein band is lost fromthe media fraction (Fig. 6A, arrows). At the same time, a simi-larly sized glycoprotein band appears in the cell layer fraction.This suggests uptake by the mineralizing cell layer, presumablyinto the BMF, because a similar glycophosphoprotein band was

shown in Fig. 2 to be specifically enriched in mineralized LCM-captured BMF.

The 75-kDa glycoprotein band is likely composed ofBAG-75 and BSP because they are the only two proteins of

this molecular weight in total bone extracts shown to reactwith digoxygenin-labeled MAA lectin (see Fig. 3H). The75-kDa phosphoprotein band is presumed to be predomi-nantlycomposed of BAG-75 because BSPfrom bone exhibitsa lowphosphate content, whereas BAG-75 contains 44 phos-

phates/mol (35). Loss from the media fraction only occurswhen mineralizationis ongoing andnot when it is blocked byinclusion of AEBSF or when BGP is omitted (Fig. 6A).Although similar analyses of thecell layer demonstratethat a

TABLE1

Effectof protease inhibitors onmineral nucleation withinbiomineralization foci inUMR106cultures

Inhibitor Target protease(s)Range of

concentration testedSpecific inhibition

of mineral deposition

AEBSF Trypsin, chymotrypsin, plasmin, thrombin, kallikrein, proprotein convertases 0.010.4 mM YesAprotinin Trypsin, chymotrypsin, and plasmin 03 g/ml NoAntipain Papain, trypsin, and plasmin 100 M NoC1s inhibitor Activated complement protein C1s 0.1100 g/ml NoE-64 Cysteine proteases 10 M No

Elastatinal Elastase and elastase-like proteases 100 M NoGM 6001 Matrix metalloproteinases 2, 3, 8, and 9 10 M NoHexa-D-arginine Furin 110 M NoHirudin Thrombin 0.510 ATUa NoLeupeptin Trypsin-like proteases and some cysteine proteases 100 M NoPefabloc PL Plasmin and plasma kallikrein 1100 M NoPefabloc urokinase-type

plasminogen activatorUrokinase plasminogen activator 1100 M No

a One antithrombin unit (ATU) will neutralize 1 NIH unit of thrombin at 37 C, based on direct comparison with an NIH thrombin reference standard.





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75-kDa glycoprotein is taken up only when mineralization isprogressing, a comparable increase in phosphoprotein (e.g.BAG-75) staining is not observed (Fig. 6A, arrows). Theseconclusions were confirmed when similar one-step extractswere probed with monospecific antibodies (Fig. 6B).Although approximately one-half of the BSP is lost from the

media fraction during mineralization (BGP), a comparableamount of BSP became associated with the cell layer.Although BAG-75 protein was also lost from the media frac-tion only when mineralization occurred (media,BGP), its

recovery in the cell layer fraction was lower than expected.This is contrary to the known presence of BAG-75 antigen inBMF and nodular complexes prior to and during their min-eralization in osteoblastic cell cultures (12). As a result, we

FIGURE 4. AEBSFinhibitsmineral nucleation bothin UMR 106 osteoblas-tic culturesand in primary mouse calvarial osteoblasts.A, with UMR cells,AEBSF blocks mineralization similarly in both serum-containing and serum-depleted conditions, while displaying higher effectiveness with mineraliza-tion competent cultures. The amounts of Alizarin red S bound to calciumphosphate crystals within BMF and cell viability assessed with the MTT assaywereplotted versus the concentrationof AEBSFadded to cultures.AEBSF waspresent twotimes duringthe culture model, e.g. from44 to64 h after plating

andfrom64 to88 h after plating. A 4-foldincrease in sensitivity wasobservedin converting from serum-sufficient conditions to serum-depleted condi-tions, whereas a 10-fold increase in effectiveness was obtained when com-paring 6488 h versus 4464 h cultures. For 6488 h cultures: , MTTabsorbance in serum-depletedconditions;f, MTTabsorbance in serum-con-taining media; E, amount of Alizarin red bound in serum-depleted condi-tions;F, amountof Alizarinred boundin serum-containing media.For44 64h cultures:, amountof Alizarin redbound in serum-containing media.(MTTassay results for 4464-h cultures were essentially identical to those for6488 h cultures and were omitted from the graph to maintain clarity.) ForAlizarin red S assays, designated results () were significantly different fromcontrols at p 0.05; for MTT cell viability assays, only cultures treated with 1mM AEBSF (*) differed significantly from controls at p 0.05. UMR culturestudies were carried outin triplicate; results shown arerepresentativeof fourseparate experiments. Error bars represent the means S.D. B, AEBSF com-pletely inhibits mineralization within nodules of primary mouse calvarial cul-tures. MTT assay results and the amount of Alizarin red S bound to mineraldeposits within cultures on day 12 are plotted versus the concentration of

AEBSF added to cultures on day 9., MTT absorbance in serum-containingmedia;E, amountof Alizarin redboundin serum-containing media.For Aliz-arinred S assays, individual datapoints () differed significantly fromcontrolsatp 0.05; for MTT cell viability assays, results (#) weresignificantly differentfromcontrols atp0.05. Primary culture studies weredone in quadruplicate;results shown are representative of three separate experiments. Error barsrepresent the mean S.D.

FIGURE 5. Phase contrast microscopy of mineralized and AEBSF-treatedprimary calvarial osteoblastic cultures. Primary calvarial osteoblastic cellswere harvested and cultured as described under Methods. On day 9 afterplating, some of theculture wells were treated with 0.01 mM AEBSF, whereascontrol cultures were re-fed normal media. Unstained cultures were photo-graphed on day 12. A, phase contrast image of control cultures. Mineralizednodules (arrows) appear as dark deposits under these conditions. B, phasecontrast image of culture treated with 0.01 mM AEBSF. No mineralized nod-uleswere visible. Results shownare representativeof multiple wellsand wereconsistent in three separate experiments. Scale bar 500m.

FIGURE 6. One-step extraction of UMRosteoblastic cell layer is unabletoaccount for the quantitative loss of BAG-75 from the media fractionoccurring during mineralization. A, extraction of cell layers with 50 mMEDTA/CHAPS extraction buffer (0.1 M Tris acetate buffer (pH 7.8), containingwith 0.5% octyl glucoside, 0.05% SDS, 0.05 M EDTA, and 0.02% sodium azide)reveals the loss of 75-kDa glycophosphoproteins from the media and thesubsequent uptake of this band into the cell layer. Treatment of the cultureswith AEBSF blocks the cell layer uptake of this same band. A, media and celllayerextractsfrom cellculturestreated with or without-glycerol phosphateand with or without AEBSF. B, extraction of cell layers with 8 M urea, 50 mMEDTA,and 0.5%SDS reveals BAG-75 is lost from themedia of-glycerol phos-

phate-treated cultures, while unaccounted for in the cell layer extract fromthe same cultures. Recovery of full-length 75-kDa BSP from the cell layer of-glycerol phosphate treated cultures, along with that for conditionedmedia, is comparable with that without -glycerol phosphate; however, noBSP fragment (4550-kDa) was detected in the cell layer fraction.





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reasoned that the one-step extraction method resulted in alower than expected recovery of BAG-75.

As an alternative, a two-step sequential extraction protocolwas used. To dissolve mineral crystals and release bound pro-teins, the cell layer was first extracted for 2 h at 4 C with 0.05 M

EDTA (pH 7.8). The residual cell layer was then treated vigor-ously with 8 M urea and 2% CHAPS (pH 7.8). Each extract wasprocessed separately and subjected to SDS-PAGE, and the gelswere stained with either Coomassie Blue dye or for glycopro-teins or phosphoproteins. Urea/CHAPS extracts showed few

differences among the four different experimental conditions(Fig. 7). In contrast, EDTA extracts of mineralizing cell layersgrown only in the presence of BGP displayed dramaticallyincreased glycoprotein- and phosphoprotein-stained bands at50 and 75 kDa when compared directly with nonmineralizing

cultures grown in the other three conditions (Fig. 7). Interest-ingly, total protein staining with Coomassie Blue showed acomparable pattern for all culture conditions suggesting theabsence of large scale proteolysis accompanying mineral nucle-ation within BMF (Fig. 7). Taken together, these findings indi-

cate that the two-step extraction method improves recoveriesof unaccounted for 75- and50-kDaglycophosphoproteins fromthecell layer of mineralized cultures.These resultsindicatethatone or more 75-kDa glycophosphoproteins present in theserum-free media compartment of UMR 106-01 cultures are

specifically taken up by the cell layer (BGP) during the min-eralization period (6488 h) (Figs. 6 and 7). Because LCM-captured BMF are highly enriched in a similar glycophospho-protein band of 75 kDa (Fig. 2), we propose that this band istaken up from the media into the cell layer where it is specifi-

cally localized within the BMF structures. When mineralizationis blocked with AEBSF, the 75-kDa glycophosphoprotein bandremains in the media fraction (Fig. 7). Likewise, in the absenceof BGP, the 75-kDa band remains in the media compartment(BGP andBGP AEBSF) (Fig. 7).

AEBSF Inhibits the Proteolytic Cleavage of BAG-75 and BSP

That Accompanies MineralizationIn view of the identifica-tion of BSP and BAG-75 as 75-kDa glycoproteins involved in

mineral nucleation and the enrich-ment of 4550-kDa fragmentswithin LCM-captured BMF (Fig. 3),

it was of interest to establishwhether their cleavage was alsosusceptible to AEBSF inhibition.UMR cultures were grown in the

presence or absence of AEBSF andof BGP. Resultant cell layer frac-tions were extracted with the two-stage extraction protocol of 0.05 M

EDTA followed by 8 M urea, 2%CHAPS (see Methods). For com-parison, all media and cell layerfractions were electrophoresed inadjacent lanes and blotted with

either MAA lectin, antibody 503(recognizes N-terminal residuesnumber 3-13 of BAG-75), anti-

body 504 (recognizes BAG-75 pro-

tein), or anti-BSP antibodies (Fig. 8, AD).Consideration of these blots revealed several interesting

points. First, full-length BAG-75 and BSP are taken up by the

cell layer only in the presence of BGP (Fig. 8, B and C). Second,

4550-kDa fragments of BAG-75 (Fig. 8A) and BSP (Fig. 8C)

were detected in the cell layer only when mineralization occurs.

Importantly, cleavage is blocked by AEBSF coincident with

inhibition of mineralization. Third, MAA lectin, which recog-

nizes both BSP and BAG-75 (Fig. 3H), also recognizes 4550-

and 75-kDa forms in mineralized cell layer fractions (Fig. 8D).

Finally, direct analyses of LCM-captured BMF have shown the

75- and 4550-kDa fragment forms of BAG-75 and of BSP are

both predominantly localized to BMF complexes (Fig. 3). Insummary, AEBSF blocks uptake and cleavage of BAG-75 and

BSP, as well as mineral nucleation within BMF.

In view of the known affinity of BSP and BAG-75 for

hydroxyapatite crystals (35, 36), it is possible that some of the

uptake by the BGP cell layer is because of direct binding to

mineral. However, a significant portion of these proteins taken

upby theBGP cell layer also occurs in the absence of mineral

and of cleavage (BGP AEBSF) (Fig. 8, B and C). Control

blots developed with MAA lectin confirm our earlier glycopro-

tein staining results showing redistribution of a 75-kDa glyco-

protein coincident with mineral crystal nucleation (Fig. 8D). In

this way, we suggest that the amount of direct protein binding

to mineral crystals is represented by the difference between the

respective 75-kDa bands in theBGP versusBGP AEBSF

lanes (Fig. 8,B and C). Although the percentage of cleaved frag-

ment relative to full-length BAG-75 or BSP in the cell layer of

mineralized cultures (BGP) is less than 50%, the absolute

amount of these stained fragments is similar to that for

uncleaved precursor proteins (Fig. 8,A and C) from nonminer-

alized cultures (BGP AEBSF). It is noteworthy that non-

mineralized cultures contain high levels of the uncleaved, full-

length protein in the media (Fig. 8, AD). Taken together, the

blotting data indicate that mineralization occurs coincident

with uptake and/or cleavage of BAG-75 and BSP by BMF.

FIGURE 7. Two-step extraction method yieldsincreased recoveries of 75- and 50-kDa glycoprotein andphosphoprotein bands. UMR cell layers were extracted first with 0.05 M EDTA and then with urea-CHAPS asdescribed under Experimental Procedures. The extracts were then processed for SDS-PAGE and the gelsstained with Pro-Q Emerald and Pro-Q Diamond fluorescent stains, or with Coomassie Blue. Compared withresults with the one-stepextraction method (Fig.6A), increasedrecoveries of 75-and 50-kDa glycoprotein andphosphoprotein bands are denoted by arrows. For reference, the appearance of relevant conditioned mediagel lanes is depicted in Fig. 6A; the conditioned media were unaffected by choice of cell layer extractionmethod.





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Blockage of this cleavage by AEBSF leads to complete inhibition

of mineral nucleation within BMF.

Two-dimensional SDS-PAGE Reveals That AEBSF Blocks theCleavage and Uptake of Other Mineralization-related Proteins

by the Cell LayerComparative analyses of EDTA extracts bySDS-PAGE (Fig. 8) prompted us to look more extensively at

whether proteins other than BAG-75 and BSP have their cleav-age inhibited by AEBSF. Cells were grown under serum-de-pleted conditions, and resultant cell layer fractions wereextracted with the two-step protocol using 0.05 M EDTA andthen 8 M urea, 2% CHAPS. Preparations from each cell layer

extract and media fraction were subjected to two-dimensionalSDS-PAGE. Gels were stained with colloidal Coomassie Blueand aligned using the PD-Quest program (Bio-Rad) to identifydifferences in the staining patterns for the BGP condition

compared with that for theBGPAEBSF condition (supplemental

Figs. 13).There were no major differences

detected between the BGP andthe BGP AEBSF-treated cul-tures for either the urea/CHAPS

extract or the media fraction (sup-plemental Figs. 2 and 3). However,the differences detected betweenthe two EDTA extracts were dra-matic (supplemental Fig. 1). Gel

spots were selected for mass spec-tral peptide mapping and liquidchromatography-tandem mass spec-trometry identification if at least a2-fold difference existed in staining

intensity between the two cultureconditions. Over 50 protein spots inEDTA fractions from AEBSF-

treated and untreated control cul-tures were identified (results not

shown). Application of the follow-ing criteria to this list identifiedthree additional AEBSF-sensitivecleavages. 1) Spot present in EDTAextract was absent in urea extract

and in media fraction. 2) Spot exhib-its substantially higher stainingintensity in the BGP condition ascompared with that in BGP

AEBSF condition. 3) Size of proteinbased on second dimension SDS-PAGE is at least 10% smaller thanexpected. 4) Apparent isoelectricpoint is inconsistent with that

expected for full-length protein.Table 2 provides a summary list

of the five proteins whose cleavageis blocked by treatment withAEBSF. These proteins are procol-

lagen C proteinase enhancer pro-tein (37), bone sialoprotein, 1,25-vi-

tamin D3

membrane-associated rapid-response steroid-binding

protein, nascent polypeptide-associated complex chain, and

bone acidic glycoprotein-75.

DISCUSSION

The data presented here support the following conclusions

about the mechanism of mineral crystal nucleation withinspherical extracellular BMF structures. First, UMR cells miner-alize equally well in the presence or absence of fetal bovineserum. Second, glycophosphoproteins BAG-75 and BSP arespecifically enriched in LCM-captured mineralized BMF as

compared with the total cell layer. Fragments of each protein(4550 kDa in apparent size) were also substantially enrichedwithin BMF. Third, a functional survey of different proteaseinhibitors showedthat AEBSF, a covalent serine protease inhib-

FIGURE 8. Western blotting demonstrates that AEBSF inhibits proteolytic cleavage of BAG-75 and BSP.UMR cultures were grown in the presence or absence of-glycerol phosphate and with or without 0.04 mMAEBSF inhibitor as described under Experimental Procedures. The conditioned media were then removed,and the cell layer was subjected to the two-step extraction method (see Fig. 7). Resultant EDTA and urea/CHAPS extracts were processed separately for immunoblotting along with the conditioned media. Immuno-staining for BAG-75 (A and B)andBSP(C) proteins andMAAlectin (D) shows thepresence offragments inEDTAextracts from mineralizing conditions only (arrows) and the loss of full-length forms from the conditionedmedia in mineralizing conditions only (arrowheads). MAA lectin binds to both full-length BAG-75and BSP(seeFig. 3. FH) and shows an enrichment of 75-kDa band only in the mineralizing conditions.





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itor, was able to specifically block mineral nucleation withinBMF in UMR 106 cultures and in mineralization nodules inprimary calvarial osteoblastic cultures. In the UMR model, the

inhibitor was 10-fold more effective if present during the min-eralization phase (64 88 h after plating) rather than during theproliferation/differentiation phase (44 64 h after plating).Similarly, in primary osteoblastic cultures, AEBSF appeared toblock a step just prior to mineral crystal nucleation. Fourth,when mineralization was blocked by AEBSF, cleavage of

BAG-75 and BSP was also inhibited. Furthermore, two-dimen-sional SDS-PAGE comparisons of UMR culture fractions in thepresence and absence of AEBSF identified three other proteins(procollagen C proteinase enhancer protein, membrane-asso-ciated rapid-response steroid receptor, and nascent polypep-

tide associated complex chain) whose cleavage was alsoblocked by this inhibitor. Fifth, although BSP and BAG-75become enriched within BMF during their mineralization, theyoriginally are also present in the media compartment. Taken

together, our results indicate for the first time that cleavage ofBAG-75, BSP, procollagenC proteinase enhancer protein, 1,25-vitamin D

3-membrane-associated rapid-response steroid-

binding protein, and nascent polypeptide associated complexchain and mineral nucleation within biomineralization foci areassociated with an AEBSF-sensitive protease produced byosteoblastic cells. The data suggest these proteolytic events maybe a permissive step for mineralization to proceed.

Selective proteolysis or fragmentation plays a critical role in

many biological processes, e.g. blood coagulation, fertilization,

and complement activation, where it represents a means to reg-ulate protein function through activation or inactivation. Ser-ine proteases play a key role in tumor-induced bone formationand tooth mineralization. Enamel mineralization requires ser-

ine proteases enamelysin (MMP-20) and kallikrein 4 (38).Enamelysin catalyzes the cleavage of amelogenin, one of themain structural proteins forming enamel. Kallikrein 4 isresponsible for degradation of enamel proteins, which results intheir removal from the matrix allowing the enamel layer to fully

mineralize. Previously,we showed stromelysincleaved BAG-75into a 50-kDa fragment in vitro (39); a similar 50-kDa fragmentis found in calcified tissue and in serum (24). Preliminary datashow that the level of 50-kDa fragment in serum correlates

directly with bone formation following ovariectomy of rats (40).BSP has also been shown to undergo proteolysis by an endoge-nous protease in UMR 106cells yielding a fragment of47 kDa

(41). This fragment is similar in size to that produced here bythe AEBSF-sensitive protease. Although cleavage of thesephos-

phoproteins hasbeen noted previously (above), our findings arethe first to link BSP and BAG-75 fragments specifically to thesite of mineral nucleation.

Because the identity of the protease responsible for endog-enous fragmentation of BSP and BAG-75 in bone is presently

unknown, we surveyed a wide range of competitive and non-competitive inhibitors against serine and cysteine proteasesand matrix metalloproteinases. Of all the inhibitors tested,AEBSF was the only one that had a specific effect on miner-

alization. Some like Pefabloc urokinase-type plasmino-gen activator were found to block mineralization nonspecifi-cally because of their associated toxicity. This was identifiedby separate, parallel assays of cell viability. For AEBSF, tox-icity was observed only at concentrations 100-fold above

those found to completely block mineral nucleation in BMF,e.g. 0.01 mM.

In addition to full-length forms, EDTA extracts of the celllayer contained a 50-kDa fragment of BAG-75 and a 45-kDa

fragment of BSP under mineralizing conditions only. A majorsource of BAG-75 and BSP was the conditioned media, becausein the absence of BGP, full-length proteins were localized pre-dominantly to the media. We have previously shown that addi-

tion of a phosphate source to 64-h cultures (mineralization-

competent) is necessary to induceBSP uptakeby BMF and theirsubsequent mineralization (12, 22); in the absence of phos-phate, BAG-75 enriched BMF remain unmineralized. The10-fold greater effectiveness of AEBSF when added to 64-h ver-

sus 44-h UMR cultures suggests that the inhibitor acts late inthe differentiation phase just prior to mineral nucleation. Sim-ilarly, in primary calvarial cultures, late addition of AEBSF onday 9 proved just as effective as on days 3 or 6 during the differ-entiation phase (5, 25, 26, 42) suggesting that AEBSF acts pref-

erentially during the period immediately before mineral crystalnucleation in nodules. In this way, both the UMR 106 and pri-mary osteoblast culture models exhibited similar sensitivities toAEBSF.

TABLE2

Proteins in EDTA extract whose fragmentation is blocked byAEBSF

Spotno.

Protein identificationObserved

massApparent mass Expected pI

Method(s) foridentification

Peptides identified(Mascot score)

Da Da

1 Procollagen C proteinaseenhancer protein

48,000 55,000 (Ref. 37) 8.5 Differential staining aftertwo-dimensional SDS-PAGEand mass spectroscopy

FDVEPDTYCR (59)TGDLDLPSPASGTSLK (49)SGTLQSNFCSSSLVVTGTVK (75)

2 1,25-D3-MARRS receptor

protein (ERp57)

50,000 57,079 5.4 Differential staining after

two-dimensional SDS-PAGEand mass spectroscopy

LNFAVASR (63)

YGVSGYPTLK (65)LAPEYEAAATR (88)FAHTNVESLVK (74)DLFSDGHSEFLK (72)

3 Nascent polypeptide associatedcomplex, chain

31,000 221,512 9.4 Differential staining of two-dimensional SDS afterPAGE and mass spectroscopy

DIELVMSQANVSR (75)SPASDTYIVFGEAK (79)NILFVITKPDVYK (80)

Bone acidic glycoprotein-75 50,000 7580,000 4.55.0 One-dimensional SDS-PAGEimmunoblotting

NAa

Bone sialoprotein 4550,000 7580,000 6.0 One-dimensional SDS-PAGEimmunoblotting

NA

a NA indicates not applicable.





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Protein uptake into the cell layer (and BMF) is a selectiveprocess because comparative one-dimensional gel analyses

revealed BGP versus BGP AEBSF cultures differed pri-marily in their content of a single 75-kDa glycophosphoproteinband shown later by immunoblotting to contain BAG-75 andBSP. Interestingly, formation of both of the 4550-kDa frag-ments was inhibited, and their incorporation into the cell layer

was blocked by inclusion of AEBSF. However, a portion of eachfull-length protein remained in the EDTA extract in BGP AEBSF cultures, suggesting that their incorporation into thecell layer required the presence of BGP and can occur prior tocleavage. Although mineral crystals are formed within BMF in

the presence of BGP, no crystals could be detected in AEBSF-treated cultures. Thus, initial binding of full-length BAG-75and BSP likely depends upon protein-protein interactionswithin the BMF complex; however, their fragmentation corre-lates with mineral crystal nucleation. This suggests that these

fragments may participate in mineral nucleation within BMF.AEBSF may be directly inhibiting a protease that is capable ofcleaving BAG-75 and BSP, or alternatively, the inhibition may

be indirect, involving multiple proteases. Further work will benecessary to identify the AEBSF-sensitive protease and to

determine whether it acts directly.This is the first report of a serine protease requirement for

bone mineral nucleation. Capable of diffusing through bilayermembranes, AEBSF is a covalent serine protease inhibitor. Ithas been used previously to block trypsin activation of pro-

tease-activated receptor in A-549 epithelial cultures (43) and inmonocyte cultures to inhibit superoxide release followingtumor necrosis factor- or platelet-activating factor stimula-

tion (44). AEBSF inactivates a wide variety of serine proteases,including chymotrypsin, urokinase plasminogen activator, kal-likrein, plasmin, thrombin, furin, and trypsin (4547). How-ever, more specific inhibitors against most of these proteaseswere unable to block mineralization. Two possible explanations

are that the effect of AEBSF may not be due to a proteolyticenzyme (AEBSF hasalso been identified as an inhibitor of phos-pholipase D (48)) or that a more specific inhibitor for theAEBSF-sensitive protease may not be available. In view of theidentification of five proteins whose cleavage is blocked by

AEBSF, the evidence strongly supports a role for a serine pro-tease in mineralization.

All five proteins whose cleavage is inhibited by AEBSF areassociated directly or indirectly in the process of bone mineral-ization. BSP is associated with mineralization in bone, teeth,

and breast cancer (4951). Both a 38-kDa mid-protein frag-ment and a 25-kDa N-terminal fragment of this protein havebeen identified as nucleators of mineralization in vitro (21).Procollagen C proteinase enhancer protein is a secreted proteinthat enhances the activity of extracellular matrix procollagen

C-terminal proteinase (BMP-1), an enzyme that activates fibril-lar assembly of type I procollagen. Procollagen C proteinaseenhancer protein knock-outmice increase thediameter of theirlong bones to apparently compensate for diminished mechan-ical performance (52). In 1997 an active fragment of this pro-

tein was identified in 3T6 fibroblast cells by Hulmes et al. (53)who suggested that cleavage of this protein was required for theformation of the extracellular matrix that will later support

mineralization. The product of procollagen C-terminal pro-teinase cleavage of procollagen I is type I collagen C-terminalpropeptide. Nicolaidou et al. (54) showed type I collagen C-ter-

minal propeptide rose following vitamin K treatment and cor-related with an increase in bone mineral density. The use of thisby-product of procollagen type I processing as a marker forbone formation(55) suggests that enhancer activation and inhi-

bition in our system may relate to its osteogenic role. Alongwith collagen type I, other substrates of procollagen C-terminalproteinase (BMP-1) include collagen type VII (55, 56) and col-lagens type II and type III (57).

1,25D3-MARRS is a membrane-associated vitamin D-bind-ing protein necessary for calcium and phosphate uptake into

the cell for support of bone development (58, 59). In 2005, Ster-ling and Nemere (60) showed that addition of an antibodyagainst 1,25D3-MARRS, or protein kinase C inhibitor calphos-tin C, inhibited vitamin D-stimulated phosphate uptake inchick intestinal cell cultures. Calcium transport has also been

shown to be regulated by vitamin D binding to 1,25D3-MARRSin aged female chicken intestine, as determined by dose-re-

sponse curves for ion transport and kinetics (61). Because1,25D3-MARRS is a plasma membrane protein, we speculatethat its presence in the EDTA cell layer extract may reflect an

association with released membrane-bound vesicles participat-ing in the process of mineral nucleation or proteolytic releasefrom the cell (46).

Finally, nascent polypeptide-associated complex chain is alarge 220-kDa cytosolic protein that translocates newly synthe-sized polypeptides to the nucleus. A C-terminal fragment ofthis protein was previously identified in an epithelial cell line

(62), and all three peptides identified in our mass spectroscopicMS/MS peptide studies were localized to this same fragment(Table 2). Identification of an intracellular protein in the EDTAextract seems counterintuitive (Table 2). However, we hypoth-esize that intracellular proteins may become entrapped within

secretory vesicles contributing to the assembly of BMF. Alter-natively, BMF formation may involve the blebbing of theplasma membrane and release of vesicular structures contain-ing selected cytoplasmic contents.

In summary, the results indicate that cleavage of BAG-75,

BSP, 1,25D3-MARRS protein, nascent polypeptide associatedcomplex chain, and procollagen propeptidase enhancer by anunidentified osteoblast-derived serine protease is associatedwith mineral nucleation. The fact that both BAG-75 and BSP

and their fragments are preferentially localized to mineralizing

BMF sites and that inhibition of their cleavage blocks mineralnucleation within BMF suggests that each plays a functionalrole in this process. Future studies will address the identity ofthe protease and effect of cleavage on the structure/function of

these proteins.

AcknowledgmentsJ. P. G. acknowledges the excellent technical

assistance of Sharon Midura and the generous assistance of Dr. Wil-

liam Landis with initial laser capture microscopy.

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