179

Journal of Oral Science, Vol. 43, No. 3, 179-188, 2001

Gene expression and immunohistochemical localization of and biglycan in association with early formation in the developing mandible

Naoko Kamiya, Kayoko Shigemasa and Minoru Takagi

Department of Anatomy and Division of Functional Morphology, Dental Research Center, Nihon University School of Dentistry, Tokyo 101-8310

(Received 13 June and accepted 27 July 2001)

Abstract: We investigated the expression of the differentiated before the onset of matrix small , decorin and biglycan, which are mineralization and that they could play a role in the associated with differentiation, and how this earliest stages of bone formation. Negative relates to the expression of osteocalcin and bone staining in the mineralized bone matrix suggests that sialoprotein (BSP) early in the formation of bone in the a loss of, or a sharp decrease in proteoglycans occurs rat mandible by immunohistochemistry and in situ concomitant with bone matrix mineralization. (J. Oral hybridization. The mandibles of rat fetuses were Sci. 43, 179-188, 2001) collected on embryonic days 14 (E14) to E18. In situ hybridization showed that expression of decorin, Key words: bone; biglycan; decorin; in situ hybri- biglycan, osteocalcin and BSP was not apparent in the dization; immunohistochemistry. developing mandible at E 14, but was expressed by newly differentiated osteoblasts at E15. The expression of these mRNAs increased linearly as the number of Introduction osteoblasts increased in specimens from E16 to E18. Bone contains a variety of noncollagenous Immunohistochemistry showed that newly differenti- such as proteoglycans, osteocalcin (bone Gla , ated osteoblasts expressed biglycan moderately, decorin BGP), matrix Gla protein (MGP), , osteopontin weakly, and osteocalcin and BSP faintly. The (OPN), bone sialoprotein (BSP), and bone acidic unmineralized bone matrices among the osteoblasts -75 (BAG-75), which are thought to be showed prominent staining for decorin, weak staining expressed during the developmental sequence of osteoblast for osteocalcin and BSP, and very weak staining for differentiation (1-6). Although the exact nature of these biglycan. When the intercellular matrix was mineralized noncollagenous proteins is not fully known, they most likely at E16, the mineralized bone matrix showed more participate in regulation of cell metabolism, matrix prominent staining for osteocalcin and BSP, but lacked deposition and mineralization as well as bone turnover (1- staining for decorin and biglycan. The same staining 6). Determination of the expression patterns of noncollagen- profile was observed during the subsequent phases of ous proteins at different stages of osteoblast differentiation bone formation at E17 and E18. These results indicate allows us to understand their functional roles in bone that decorin, biglycan, osteocalcin and BSP are formation. expressed at the gene and protein level by newly The proteoglycans that have been identified in bone matrices are mainly of low-molecular-weight with one or two chains (1-4). Two members of this Correspondence to Dr. Minoru Takagi, Department of Anatomy, Nihon University School of Dentistry, 1-8-13, Kanda-Surugadai, group, PG I (biglycan) and PG II (decorin), are different Chiyoda-ku, Tokyo 101-8310, Japan gene products (7). Decorin is also termed PG-SII (8-10). Tel: +81-3-3219-8110 Fax: +81-3-3219-8318 Synthesis and secretion of these two proteoglycans by E-mail address: takagi-mn@ dent.nihon-u.ac.jp osteoblasts have been confirmed by in vitro studies using 180

osteoblastic cell cultures (11,12). The specimens were dehydrated in a graded ethanol series

Previous immunohistochemical studies of the and embedded in paraffin wax. Sections, 6 ƒÊm thick, were metaphyseal bone trabeculae of bovine fetus utilizing an dewaxed in xylene, and rehydrated through descending antibody specific for small proteoglycans concentrations of ethanol. Some sections were stained have shown that the antibody stains osteoblasts and osteoid with either hematoxylin and eosin or alizarin red S stain but not the mineralized bone matrix (13). Bianco et al. (22), and others were either hybridized or immunostained

(14,15) have demonstrated both decorin and biglycan in as described below. osteoblasts and osteoid at all sites of endochondral and membranous ossification, whereas mineralized bone Preparation of RNA probes for decorin, biglycan, matrices were not reactive. In situ hybridization studies osteocalcin and BSP have also demonstrated mRNA expression of biglycan The pGEM4Z plasmid containing rat biglycan cDNA and decorin in osteoblasts (14-16). However, these studies (23) kindly provided by Dr. K. L. Dreher (Pulmonary have not examined the expression of decorin and biglycan Toxicology Branch, Experimental Toxicology Division, mRNAs associated with osteoblast differentiation, and a USPA, NHEERL, Research Triangle Park, NC, USA) was correlation between both small proteoglycans and other digested with Pstl and Smal to obtain a 453-bp biglycan bone matrix proteins. cDNA fragment (nucleotides 544-996), which was then

This study has utilized in situ hybridization and subcloned into the pGEM3Zf(+)(Promega). The immunohistochemistry to investigate the expression of pGEM3Zf(+) plasmid containing rat decorin cDNA (24), the small proteoglycans, decorin and biglycan, which are kindly provided by Dr. S. Abramson (Cleveland Clinic associated with osteoblast differentiation, and how this Florida, Miami, FL, USA), was digested with Hindi and relates to the expression of osteocalcin and BSP in early self-ligated. The resulting plasmid contained an 811-bp bone formation of the developing rat mandible. decorin cDNA fragment (nucleotides 501-1311). The

pT7/T3-18 plasmid containing rat bone sialoprotein cDNA

Materials and Methods (25,26), kindly provided by Dr. Y. Ogata (Department of Tissue preparation Periodontology, Nihon University School of Dentistry at

Developing mandibles were collected from the fetuses Matsudo), was digested with Hindi and self-ligated. The of Wistar rats (n=15) at embryonic days 14 (E14) to El8 resulting plasmid contained a 429-bp BSP cDNA fragment under Nembutal anesthesia. The specimens used for in situ (nucleotides 611-1039). hybridization were immersed in 4% paraformaldehyde A rat osteocalcin cDNA fragment (27) was obtained by fixative in 0.1 M cacodylate buffer (pH 7.3) for 18 h at reverse transcriptase polymerase chain reaction (RT-PCR)

4•Ž. The specimens used for light microscopic of total RNA from 2-day-old rat mandibular bone using immunohistochemistry were immersed in modified the following primers: 5'-GAA CAG ACA AGT CCC

Karnovsky's (17) fixative containing 4% paraformaldehyde- ACA C-3' (nucleotide 1-19) and 5'-CTA AAC GGT GGT

0.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) GCC ATA G-3'(nucleotide 330-348). The PCR product was in the absence or presence of 0.3% cetylpyridinium chloride subcloned into pT7BlueT vector (Novagen) and digested

(to retain soluble proteoglycans (18)) for 18 h. After with Pstl and Sad to obtain a 270-bp osteocalcin cDNA fixation, the specimens were rinsed thoroughly in 0.1 M fragment (nucleotides 1-270), then it was further subcloned cacodylate buffer (pH 7.3) at 4•Ž. Some specimens were into pGEM3Zf(+). left undemineralized, and other specimens were To synthesize the antisense or sense probe, plasmids were demineralized for two weeks at 4•Ž in 0.2 M triethyl- linearized with HindIII or EcoRI. Digoxigenin (DIG)- ammonium EDTA solution in absolute ethanol diluted labeled antisense or sense probes were synthesized using

4:1 in distilled water (ethanolic alkylammonium EDTA) DIG-11-uridine-5'-triphosphate (DIG-UTP; Roche according to Scott and Kyffin (19). The use of ethanolic Diagnostics GmbH, Mannheim, Germany) in T7, T3 or alkylammonium EDTA for demineralization of aldehyde- SP6-primed RNA polymerase reaction. fixed bone specimens results in good retention of proteoglycans and in bone tissues (20,21) In situ hybridization without reducing antigenicity (21). The specimens with Deparaffinized sections were rinsed with PBS, treated and without EDTA demineralization were processed for with proteinase K (Life Technologies, Inc., Rockville, paraffin embedding. MD, USA), 2 ƒÊg/ml in PBS for 30 min at 37•Ž, washed in PBS, and treated with 4% paraformaldehyde in PBS for

Light microscopy 10 min at room temperature. The sections were washed 181

in PBS, then treated with 0.2 N HCl, and washed again kindly provided by Dr. Larry W. Fisher, National Institute with PBS. The sections were rinsed thoroughly with PBS, of Dental Research, NIH, MD, USA. The goat anti-rat acetylated in 0.25% acetic anhydride in 0.1 M osteocalcin antibody (BT-413) was obtained commercially triethanolamine, pH 8.0, for 10 min at room temperature, from Biomedical Technologies Inc. Stoughton, MA, USA and washed in PBS before and after treatment of the and proved to be specific for the intact molecule of rat sections with glycine, 2 mg/ml in PBS for 30 min at room osteocalcin with the recognition site apparently involving temperature (28). The samples were dehydrated in the carboxyl terminal. The rabbit anti-porcine BSP antibody ascending concentrations of ethanol and air-dried. The was kindly provided by Dr. Jaro Sodek, University of hybridization mixture consisted of 50% formamide, 10 mM Toronto, Ontario, Canada and its specificity is described

Tris-HCl (pH 7.6), 0.2 mg/ml yeast tRNA (Roche elsewhere in detail (30).

Diagnostics GmbH, Mannheim, Germany), 600 mM NaCl, Tissue sections for immunohistochemistry were rinsed

0.25% sodium dodecyl sulfate (SDS), 1 x Denhardt's thoroughly in PBS, pH 7.3, at room temperature before solution, 10% dextran sulphate, and 300 ng/ml DIG- and after treatment with 0.1% glycine in PBS for 30 min labeled RNA probe. The tissue sections were prehybridized to quench free aldehyde groups (28). When either LF-113 in the hybridization solution without the DIG-labeled or LF-106 is used, prior digestion with specific glycosidase

RNA probe for 60 min and then in the complete is not required. However, we evaluated immunostaining hybridization mixture for 16 h at 48•Ž in a humid after pre-treatment with chondroitinase ABC, since this atmosphere. The tissue sections were rinsed with 2 x SSC, increases signal strength by elimination of chondroitinase then 50% formamide for 30 min at 48•Ž, and treated with ABC-digestible (29). The sections

TNE solution (10 mM Tris-HCl, 500 mM NaCl, 1 mM were washed briefly in 0.1 M sodium acetate-0.1 M Tris-

EDTA, pH 7.6) containing 20ƒÊg/mlRNase A (Boehringer- HCl buffer (pH 7.3) and exposed for 60 min at 37•Ž to a

Mannheim GmbH). The tissue sections were washed with protease-free chondroitinase ABC (from Proteus vulgaris) TNE solution, then sequentially washed in 2 •~ SSC, 1 •~ solution (0.2 U/ml in 0.1 M Tris-acetate, pH 7.3, containing

SSC and 0.2 •~ SSC for 30 min each at 48•Ž. The tissue 1 mg/ml BSA) (31). The enzyme was obtained from sections were incubated in DIG buffer 1 (100 mM Tris- Seikagaku Corporation, Tokyo, Japan. All subsequent

HCl, 150 mM NaCl, pH 7.6) for 5 min at room temperature steps were carried out in a moisture chamber at room and then DIG buffer 1 containing 1.5% blocking reagent temperature.

(Roche Diagnostics GmbH, Mannheim, Germany) and The tissue sections, with or without chondroitinase 1.5% bovine serum albumin (BSA) for 60 min at room ABC pre-treatment were treated with the PBS solution temperature. The tissue sections were incubated in an containing 0.3% H2O2 and 0.1% sodium azide for 30 min alkaline phosphatase-conjugated Fab fragment of sheep to eliminate endogenous peroxidase activity (32). The anti-DIG (Roche Diagnostics GmbH, Mannheim, sections were rinsed thoroughly in PBS and exposed for

Germany) diluted 1:500 in DIG buffer 1 containing 1.5% 60 min to the PBS solution, to which BSA was added at blocking reagent (Roche Diagnostics GmbH, Mannheim, a concentration of 1% (PBS and BSA). The sections were

Germany) and 1.5% BSA for 30 min at room temperature. rinsed briefly with PBS and BSA containing either 1%

The sections were washed thoroughly in DIG buffer 1, and normal goat serum or normal rabbit serum, then treated incubated in 50 mM MgCl2 , 100 mM Tris-HCl, 100 mM separately for 60 min with the following antibodies: rabbit

NaCl, pH 9.5 for 3 min at room temperature. The tissue anti-rat decorin, rabbit anti-rat biglycan (1:600 dilution), sections were treated in the incubation medium (Roche goat anti-rat osteocalcin (1:200 dilution) or rabbit anti-

Diagnostics GmbH, Mannheim, Germany) consisting of porcine BSP (1:400 dilution) in PBS and BSA containing nitro blue tetrazolium chloride (NBT) and 5-bromo-4- either 1% normal goat serum or rabbit serum. The chloro-3-indolyl phosphate, toluidine salt (BCIP) diluted specimens were then rinsed in PBS and BSA and exposed

1: 50 in 50 mM MgCl2, 100 mM Tris-HCl, 100 mM NaCl, for 10 min to one of the biotin-conjugated secondary pH 9.5 for 3 h at room temperature, stopped in 10 mM Tris- antibodies either affinity purified F(ab')2 of goat anti- HCl, 1 mM EDTA, pH 7.6 and mounted in embedding rabbit IgG (1:400 dilution) or affinity purified F(ab')2 of medium. rabbit anti-goat IgG (1:200 dilution) (Zymed Laboratories,

Inc., San Francisco, CA, USA) in PBS and BSA. The

Light microscope immunohistochemistry specimens were then rinsed in PBS and BSA and exposed

The specificity of the rabbit antisera against either rat for 15 min to horseradish-peroxidase-conjugated decorin (LF-113) or rat biglycan (LF-106) used in this study streptavidin (1:500 dilution) (Biosource International, was described elsewhere in detail (29). Both antibodies were Tagoimmunologicals, Camarillo, CA, USA) in PBS and 182

BSA. They were rinsed thoroughly with PBS and BSA, at E17 and E 18, resulting in more mineralized bone matrix and incubated in 3,3'-diaminobenzidine tetrahydrochloride (not illustrated). (DAB; Sigma Chemical Co., MO, USA) substrate solution without H2O2 for 20 min and the complete DAB substrate In situ hybridization solution for 10 min as described previously (33). The Gene expression of decorin, biglycan, osteocalcin and substrate medium consisted of 100 ml of 0.05 M Tris-HCl BSP was not detected in the developing mandible at E 14 buffer (pH 7.6), in which 50 mg of DAB was dissolved (not illustrated), but was expressed by newly differentiated and 0.1 ml of 5.2% H2O2 was added immediately before osteoblasts at E15 (Figs. 3-6). These osteoblasts expressed use. Tissue sections were rinsed with 0.05 M Tris-HCl decorin, biglycan and osteocalcin weakly and BSP strongly. buffer, dehydrated with ethanol, cleared in xylene, and The expression of these mRNAs increased linearly as the mounted for light microscope examination. For control number of osteoblasts in the specimens increased between preparations, either normal rabbit serum or normal goat El 6 (not illustrated) and E 18 (Figs. 7-10). In control serum was substituted for the primary antibodies. sections, hybridized with the sense probes, no positive labeling was seen (not illustrated). Results Light microscopy Immunohistochemistry At embryonic day 14 (E14), osteoprogenitor cells Immunostaining for decorin, biglycan, osteocalcin and accumulated at centers of ossification in close proximity BSP was not observed in the developing mandible to developing Meckel's , which appeared to consist specimens at El 4 (not illustrated). At E15, newly of condensation of the ectomesenchymal cells or differentiated osteoblasts expressed decorin weakly, chondroblasts (not illustrated). At E15, initiation of biglycan moderately, and osteocalcin and BSP faintly mandibular bone formation was observed with concurrently (Figs. 11-14). The unmineralized bone matrix among the the appearance of osteoblasts, which were newly osteoblasts showed prominent staining for decorin, weak differentiated from osteoprogenitor cells (Fig. 1). A distinct staining for osteocalcin and BSP, and very weak staining intercellular unmineralized matrix was observed among for biglycan (Figs. 11-14). At E16, concomitant with these osteoblasts. At E16, alizarin red S staining for matrix mineralization, the intercellular mineralized bone calcium was identified in the intercellular matrix among matrix appeared to lack staining for decorin and biglycan, the osteoblasts (Fig. 2). Further bone formation continued but there was distinct staining for osteocalcin and BSP. The

Fig. 1 Early-developing mandible from rat fetuses at E15, stained with haematoxylin and eosin. Initiation of mandibular bone

formation is observed with the appearance of osteoblasts (Ob; and enlarged in b) in the close vicinity of Meckel's cartilage

(M). The intercellular unmineralized matrix (arrowheads) among osteoblasts is evident. (a) •~140; (b) •~270

Fig. 2 Paraffin sections of early developing mandible from rat fetuses at E 16 show alizarin red S staining. Mineralized bone

matrices (arrows) are positively stained with alizarin red S. (a) •~140; (b) •~270 183

unmineralized bone matrix, osteoid showed strong serum or normal goat serum instead of the primary antibody immunostaining for decorin, but undetectable levels of (not illustrated). biglycan, osteocalcin and BSP (Figs. 15-18). A similar immunoreactivity was observed in the mineralized sites, Discussion irrespective of whether specimens were pretreated before This study utilized in situ hybridization and immuno- immunostaining with ethanolic alkylammonium EDTA (not histochemistry to investigate the expression of the small illustrated). The same staining profile was observed during proteoglycans decorin and biglycan, which are associated subsequent phases of bone formation at E17 (not illustrated) with osteoblast differentiation, and how this relates to the and E18 (Figs. 19-22). No reactivity was observed in any expression of osteocalcin and BSP in early bone formation of the controls that were exposed to either normal rabbit of the developing rat mandible. In situ hybridization and

Figs. 3-6 Gene expression of decorin (Fig. 3), biglycan (Fig. 4), osteocalcin (Fig. 5) and BSP (Fig. 6) in early developing mandible from rat fetuses at E15 using in situ hybridization. Osteoblasts show prominent mRNA expression of BSP and less intense but distinct mRNA expression of decorin, biglycan and osteocalcin. (a) •~140; (b) •~270

Figs. 7-10 Gene expression of decorin (Fig. 7), biglycan (Fig. 8), osteocalcin (Fig. 9) and BSP (Fig. 10) in developing mandible from rat fetuses at E18 using in situ hybridization. Osteoblasts demonstrate increased mRNA expression of decorin,

biglycan, osteocalcin and BSP compared to those of specimens at E15. (a) •~140; (b) •~270 184

immunohistochemistry in the present study have secretion of decorin into the unmineralized bone matrix demonstrated that the mRNAs and core proteins of decorin predominates over that of biglycan. This is supported by and biglycan as well as those of osteocalcin and BSP were previous studies that have shown that decorin is exclusively first expressed by osteoblasts newly differentiated from a matrix molecule and may function in regulating osteoprogenitor cells in the developing rat mandible at EIS. fibril formation, modulating transforming The differentiated osteoblasts were closely associated β (TGF-β) action, and cell growth (34,35). Biglycan is not with the unmineralized bone matrix, which showed exclusively a matrix molecule and it may be involved prominent immunostaining for decorin, but very weak directly in cell-regulatory functions, including cell immunostaining for biglycan, suggesting that osteoblasts proliferation and differentiation (14,15). synthesizes both proteoglycans, but the extracellular Differentiating and mature osteoblasts are known to

Figs. 11-14 Immunostaining for decorin (Fig. 11), biglycan (Fig. 12), osteocalcin (Fig. 13) and BSP (Fig. 14) in early developing mandible from rat fetuses at E15. The unmineralized bone matrix (arrowheads) among osteoblasts demonstrates prominent staining for decorin and BSP, weak staining for osteocalcin and very weak staining for biglycan. Biglycan staining

appears to be confined mostly to osteoblasts. (a) •~140; (b) •~270

Figs. 15-18 When matrix mineralization initiates at E16, the mineralized bone matrix (arrows) does not reveal distinct

immunostaining for decorin (Fig. 15) and biglycan (Fig. 16), but there is marked immunostaining for osteocalcin

(Fig. 17) and BSP (Fig. 18). Staining for decorin is observed prominently in the unmineralized bone matrix osteoid (arrowhead). (a) •~140; (b) •~270 185 express and a variety of noncollagenous mineralized bone tissues. Our observation indicates that proteins (36-38). Previous in situ hybridization studies osteocalcin and BSP are incorporated into the mineralized have demonstrated mRNA expression of osteocalcin (39- bone matrix and become specific components of bone 43) and BSP (40,43,44) in osteoblasts associated with tissues. developing bone tissues. Some of these have indicated that On the other hand, the failure to identify positive osteocalcin mRNA is expressed at late stages of osteoblast immunostaining for decorin and biglycan in the mineralized differentiation in the process of bone mineralization and bone matrix is similar to the situation in immuno- maturation (42), whereas BSP mRNA is expressed by histochemical studies of human and bovine bone (13-15). osteoblasts earlier than osteocalcin mRNA (43). Our study However, previous studies have demonstrated prominent shows that the mRNAs of OC and BSP, similar to those proteoglycan immunostaining in the walls of osteocyte of decorin and biglycan, are expressed almost simul- lacunae and bone canaliculi in human, rat and rabbit bone taneously by newly differentiated osteoblasts before the tissues (14,15,57,58), suggesting that the stainable onset of bone matrix mineralization. proteoglycans in this specialized region inhibit expansion It has been reported that in the immunohistochemical of matrix mineralization beyond this border and facilitate identification of noncollagenous proteins in mineralized retention of the lacunocanalicular system (57). Negative bone matrix, either masking or inability to stain proteoglycan staining in the mineralized bone matrix noncollagenous bone matrix proteins with hydroxyapatite suggests that a loss of, or a sharp decrease in proteoglycans occurs at mineralized sites (45). In this study, immuno- occurs concomitant with bone matrix mineralization as localization of the small proteoglycans, osteocalcin and indicated previously (59-66), but does not rule out BSP was identified in sections with or without involvement of these small proteoglycans in this process. demineralization with ethanolic trimethylammonium In fact, in vivo studies examining incorporation of EDTA (19), which is known to retain water-soluble [35S] sulfate into glycosaminoglycans by mineralized tissues proteoglycans in the matrix during demineralization (20,21). have shown that a population of proteoglycans migrate Following initiation of bone matrix mineralization at E16, rapidly to the mineralized bone (67). Further detailed mineralized bone matrix, with or without demineralization, studies are necessary to clarify the contribution of small showed significant immunostaining for osteocalcin and proteoglycans to bone mineralization. BSP, but no immunostaining for the small proteoglycans. The former observation is consistent with previous light Acknowledgments and electron microscopic immunohistochemical studies to This work was supported by Uemura Fund, Nihon localize osteocalcin (46-54) and BSP (30,44,55,56) in the University School of Dentistry and a Grant from the

Figs. 19-22 At E18, the mineralized bone matrix (arrows) appears to be not reactive for decorin (Fig. 19) and biglycan (Fig. 20), but there is increased immunostaining for osteocalcin (Fig. 21) and BSP (Fig. 22) when compared to that at E16.

Although strong staining for decorin is visible in the unmineralized bone matrix (arrowheads), staining for biglycan, osteocalcin and BSP is not observed clearly at this site. (a) •~140; (b) •~270 186

Ministry of Education, Culture, Sports, Science, and Rosenberg, L.C. (1986) Localization of a dermatan

Technology to promote multi-disciplinary research projects. sulfate proteoglycan (DS-PGII) in cartilage and the presence of an immunologically related species in References other tissues. J. Histochem. Cytochem. 34, 619-

1. Butler, W.T. (1984) Matrix macromolecules of bone 625

and dentin. Coll. Relat. Res. 4, 297-307 14. Bianco. P., Fisher, L.W., Young, M.F., Termine,

2. Fisher, L.W. (1985) The nature of the proteoglycans J.D. and Gehron Robey, P. (1990) Expression and

of bone. In The chemistry and biology of mineralized localization of the two small proteoglycans biglycan

tissues. Butler, W.T. ed., Ebsco Media, Birmingham, and decorin in developing human skeletal and non- 188-196 skeletal tissues. J. Histochem. Cytochem. 38, 1549-

3. Fisher, L.W. and Termine, J.D. (1985) 1563 Noncollagenous proteins influencing the local 15. Bianco, P., Riminucci, M. and Fisher, L.W. (1993)

mechanisms of calcification. Clin. Orthop. 200, Biglycan and decorin in intact developing tissues: 362-385 the in situ approach to their role in development,

4. Boskey, A.L. (1989) Noncollagenous matrix proteins morphogenesis and tissue organization. In Dermatan and their role in mineralization. Bone Miner. 6, sulfate proteoglycans. Chemistry, biology, chemical 111-123 pathology, Scott, J.E. ed., Portland Press, London, 5. Heinegard, D., Hultenby, K., Oldberg,A., Reinholt, 193-205 F. and Wendel, M. (1989) Macromolecules in bone 16. Mundlos, S. and Zabel, B. (1994) Developmental matrix. Connect. Tissue Res. 21, 3-11 expression of human cartilage matrix protein. Dev. 6. Butler, W.T. (1991) Sialoproteins of bone and dentin. Dyn. 199, 241-252

J. Biol. Buccale 19, 83-89 17. Karnovsky, M.J. (1961) A formaldehyde- 7. Fisher, L.W., Termine, J.D. and Young, M.F. (1989) glutaraldehyde fixative of high osmolarity for use Deduced protein sequence of bone small proteo- in electron microscopy. J. Cell Biol. 27, 137A

glycan I (biglycan) shows homology with proteo- 18. Kupchella, C.E., Matsuoka, L.Y., Bryan, B.,

glycan II (decorin) and several nonconnective tissue Wortsman, J. and Dietrich, J.G. (1984) Histochemical

proteins in a variety of species. J. Biol. Chem. 264, evaluation of glycosaminoglycan deposition in the 4571-4576 . J. Histochem. Cytochem. 32, 1121-1124 8. Franzen, A. and Heinegard, D. (1984) Extraction and 19. Scott, J.E. and Kyffin, T.W. (1978) Demineralization

purification of proteoglycans from mature bovine in organic solvents by alkylammonium salts of bone. Biochem. J. 224, 47-58 ethylenediaminetetra-acetic acid. Biochem. J. 169, 9. Franzen, A. and Heinegard D. (1984) 697-701 Characterization of proteoglycans from the calcified 20. Dickson, I.R. and Jande, S.S. (1980) Effects of matrix of bovine bone. Biochem. J. 224, 59-66 demineralization in an ethanolic solution of 10. Heinegard, D., Bjorne-Persson, A., Coster, L., triethylammonium EDTA on solubility of bone Franzen, A., Gardell, S., Malmstrom, A., Paulsson, matrix components and on ultrastructural M., Sandfalk, R. and Vogel, K. (1985) The core preservation. Calcif. Tissue Int. 32, 175-179

proteins of large and small interstitial proteoglycans 21. Takagi, M., Maeno, M., Takahashi, Y. and Otsuka, from various connective tissues form distinct K. (1992) Biochemical and immuno- and lectin- subgroups. Biochem. J. 230, 181-194 histochemical studies of solubility and retention of 11. Breuer, B., Schmidt, G. and Kresse, H. (1990) Non- bone matrix proteins during EDTA demineralization. uniform influence of transforming growth factor-ƒÀ Histochem. J. 24, 78-85 on the biosynthesis of different forms of small 22. Lillie, R.D. and Fullmer, H.M. (1976) chondroitin sulphate/dermatan sulphate proteo- Histopathologic technique and practical histo-

glycan. Biochem. J. 269, 551-554 chemistry. 4th ed., McGraw-Hill, New York, 539- 12. Takeuchi, Y., Fukumoto, S. and Matsumoto, T. 540

(1995) Relationship between actions of transforming 23. Dreher, K.L., Asundi, V., Matzura, D. and Cowan,

growth factor (TGF)-ƒÀ and cell surface expression K. (1990) Vascular smooth muscle biglycan of its receptors in clonal osteoblastic cells. J. Cell. represents a highly conserved proteoglycan within Physiol. 162, 315-321 the arterial wall. Eur. J. Cell Biol. 53, 296-304 13. Poole, A.R., Webber, C., Pidoux, I., Choi, H. and 24. Abramson, S.R. and Woessner, J.F. Jr. (1992) cDNA 187

sequence for rat dermatan sulfate proteoglycan-II 36. Yoon, K., Buenaga, R. and Rodan, G.A. (1987) (decorin). Biochim. Biophys. Acta 1132, 225-227 Tissue specificity and developmental expression of 25. Ogata, Y., Yamauchi, M., Kim, R.H., Li. J.J., rat osteopontin. Biochem. Biophys. Res. Commun. Freedman, L.P. and Sodek, J. (1995) Glucocorticoid 148, 1129-1136 regulation of bone sialoprotein (BSP) gene 37. Owen, T.A., Aronow, M., Shalhoub, V., Barone, expression. Identification of a glucocorticoid L.M., Wilming, L., Tassinari, M.S., Kennedy, M.B., response element in the bone sialoprotein gene Pockwinse, S., Lian, J.B and Stein, G.S. (1990) promoter. Eur. J. Biochem. 230, 183-192 Progressive development of the rat osteoblast 26. Ogata, Y., Niisato, N., Furuyama, S., Cheifetz, S., phenotype in vitro: reciprocal relationships in Kim, R.H., Sugiya, H. and Sodek, J. (1997) expression of associated with osteoblast Transforming growth factor-beta 1 regulation of proliferation and differentiation during formation of bone sialoprotein gene transcription: identification the bone extracellular matrix. J. Cell. Physiol. 143, of a TGF-beta activation element in the rat BSP gene 420-430 promoter. J. Cell. Biochem. 65, 501-512 38. Gehron Robey, P., Bianco, P. and Termine, J.D. 27. Celeste, A.J., Rosen, V., Buecker, J.L., Kriz, R., (1992) The cellular biology and molecular Wang, E.A. and Wozney, J.M. (1986) Isolation of biochemistry of bone formation. In Disorders of the human gene for bone gla protein utilizing mouse bone mineral metabolism, Coe, F.L. and Favus, M.J. and rat cDNA clones. EMBO J. 5, 1885-1890 eds., Raven Press, New York, 241-263 28. Tokuyasu, K.T. (1986) Application of 39. Weinreb, M., Shinar, D. and Rodan, G.A. (1990) cryoultramicrotomy to immunocytochemistry. J. Different pattern of alkaline phosphatase, Microsc. 143, 139-149 osteopontin, and osteocalcin expression in 29. Fisher, L.W., Stubbs, J.T. III. and Young, M.F. developing rat bone visualized by in situ (1995) Antisera and cDNA probes to human and hybridization. J. Bone Miner. Res. 5, 831-842 certain animal model bone matrix noncollagenous 40. Chen, J., Shapiro, H.S. and Sodek, J. (1992) proteins. Acta Orthop. Scand. Suppl. 266, 61-65 Developmental expression of bone sialoprotein 30. Chen, J., Zhang, Q., McCulloch, C.A. and Sodek, mRNA in rat mineralized connective tissues. J. J. (1991) Immunohistochemical localization of bone Bone Miner. Res. 7, 987-997 sialoprotein in foetal porcine bone tissues: 41. Ikeda, T., Nomura, S., Yamaguchi, A., Suda, T. and comparisons with secreted phosphoprotein 1 (SPP- Yoshiki, S. (1992) In situ hybridization of bone 1, osteopontin) and SPARC (osteonectin). matrix proteins in undecalcified adult rat bone Histochem. J. 23, 281-289 sections. J. Histochem. Cytochem. 40, 1079-1088 31. Couchman, J.R., Caterson, B., Christner, J.E. and 42. Nakase, T., Takaoka, K., Hirakawa, K., Hirota, S., Baker, J.R. (1984) Mapping by monoclonal antibody Takemura, T., Onoue, H., Takebayashi, K., Kitamura, detection of glycosaminoglycans in connective Y. and Nomura, S. (1994) Alterations in the tissues. Nature 307, 650-652 expression of osteonectin, osteopontin and 32. Li, C.Y., Ziesmer, S.C. and Lazcano-Villareal, O. osteocalcin mRNAs during the development of (1987) Use of azide and hydrogen peroxide as an skeletal tissues in vivo. Bone Miner. 26, 109-122 inhibitor for endogenous peroxidase in the 43. Sommer, B., Bickel, M., Hofstetter, W. and immunoperoxidase method. J. Histochem. Wetterwald, A. (1996) Expression of matrix proteins Cytochem. 35, 1457-1460 during the development of mineralized tissues. Bone 33. Graham, R.C. Jr. and Karnovsky, M.J. (1966) The 19, 371-380 early stages of absorption of injected horseradish 44. Bianco, P., Fisher, L.W., Young, M.F., Termine, peroxidase in the proximal tubules of mouse kidney: J.D. and Gehron Robey, P. (1991) Expression of bone ultrastructural cytochemistry by a new technique. sialoprotein (BSP) in developing human tissues. J. Histochem. Cytochem. 14, 291-302 Calcif. Tissue Int. 49, 421-426 34. Yamaguchi, Y., Mann, D.M. and Ruoslahti, E. (1990) 45. Kumagai, T., Lee, I., Ono, Y., Maeno, M. and Takagi, Negative regulation of transforming growth factor- M. (1998) Ultrastructural localization and βby the proteoglycan decorin. Nature 346, 281-284 biochemical characterization of in 35. Ruoslahti, E. and Yamaguchi, Y. (1991) developing rat bone. Histochem. J. 30, 111-119 Proteoglycans as modulators of growth factor 46. Bianco, P., Hayashi, Y., Silvertrini, G., Termine, activities. Cell 64, 867-869 J.D. and Bonucci, E. (1985) Osteonectin and Gla- 188

protein in calf bone: ultrastructural immuno- 56. Ingram, R.T., Clarke, B.L., Fisher, L.W. and histochemical localization using the protein A-gold Fitzpatrick, L.A. (1993) Distribution of noncollagen-

method. Calcif. Tissue Int. 37, 684-686 ous proteins in the matrix of adult human bone: 47. Bronckers, A.L., Gay, S., Dimuzio, M.T. and Butler, evidence of anatomic and functional heterogeneity.

W.T. (1985) Immunolocalization of 7 -carboxy- J. Bone Miner. Res. 8, 1019-1029

glutamic acid containing proteins in developing rat 57. Takagi, M., Maeno, M., Kagami, A., Takahashi, Y. . Coll. Relat. Res. 5, 273-281 and Otsuka, K. (1991) Biochemical and

48. Bronckers, A.L., Gay, S., Finkelman, R.D. and immunocytochemical characterization of mineral Butler, W.T. (1987) Developmental appearance of binding proteoglycans in rat bone. J. Histochem.

Gla proteins (osteocalcin) and alkaline phosphatase Cytochem. 39, 41-50 in tooth germs and bones of the rat. Bone Miner. 2, 58. Takagi, M., Maeno, M., Yamada, T., Miyashita, K. 361-373 and Otsuka, K. (1996) Nature and distribution of

49. Groot, C.G., Danes, J.K., Blok, J., Hoogendijk, A. chondroitin sulphate and dermatan sulphate and Hauschka, P.V. (1986) Light and electron proteoglycans in rabbit alveolar bone. Histochem. microscopic demonstration of osteocalcin J. 28, 341-351 antigenicity in embryonic and adult rat bone. Bone 59. Pugliarello, M.C., Vittur, F., De Bernard, B., Bonucci,

7, 379-385 E. and Ascenzi, A. (1970) Chemical modifications 50. Camarda, A.J., Butler, W.T., Finkelman, R.D. and in osteones during calcification. Calcif. Tissue Res. Nanci, A. (1987) Immunocytochemical localization 5, 108-114 of ƒÁ -carboxyglutamic acid-containing proteins 60. Baylink, D., Wergedal, J. and Thompson, E. (1972)

(osteocalcin) in rat bone and dentin. Calcif. Tissue Loss of proteinpolysaccharides at sites where bone Int. 40, 349-355 mineralization is initiated. J. Histochem. Cytochem. 51. Mark, M.P., Prince, C.W., Gay, S., Austin, R.L., 20, 279-292

Bhown, M., Finkelman, R.D. and Butler, W.T. (1987) 61. Wergedal, J.E. and Baylink, D.J. (1974) Electron A comparative immunocytochemical study on the microprobe measurements of bone mineralization subcellular disributions of 44 kDa bone rate in vivo. Am. J. Physiol. 226, 345-352

phosphoprotein and bone y-carboxyglutamic acid 62. Prince, C.W., Rahemtulla, F. and Butler, W.T. (1983) (Gla)-containing protein in osteoblasts. J. Bone Metabolism of rat bone proteoglycans in vivo. Miner. Res. 2, 337-346 Biochem. J. 216, 589-596 52. Mark, M.P., Butler, W.T., Prince, C.W., Finkelman, 63. Takagi, M., Parmley, R.T., Toda, Y. and Denys, F.R. R.D. and Ruch, J-V. (1988) Developmental (1983) Ultrastructural cytochemistry of complex expression of 44-k Da bone phosphoprotein carbohydrates in osteblasts, osteoid, and bone matrix.

(osteopontin) and bone ƒÁ -carboxyglutamic acid Calcif. Tissue Int. 35, 309-319

(Gla)-containing protein (osteocalcin) in calcifying 64. Kazama, T., Takagi, M., Ishii, T. and Toda, Y. (1992) tissues of rat. Differentiation 37, 123-136 Immunoelectron microscopic studies of glyco- 53. Vermeulen, A.H.M., Vermeer, C. and Bosman, F.T. saminoglycans in the metaphyseal bone trabeculae

(1989) Histochemical detection of osteocalcin in of growing rats. Histochem. J. 24, 747-755 normal and pathological human bone. J. Histochem. 65. Takeuchi, Y., Matsumoto, T., Ogata, E. and Shishiba, Cytochem. 37, 1503-1508 Y. (1990) Isolation and characterization of 54. Ohta, T., Mori, M., Ogawa, K., Matsuyama, T. and proteoglycans synthesized by mouse osteoblastic Ishii, S. (1989) Immunocytochemical localization cells in culture during the mineralization process. of BGP in human bones in various developmental Biochem. J. 266, 15-24 stages and pathological conditions. Virchows Arch. 66. Hoshi, K., Kemmotsu, S., Takeuchi, Y., Amizuka, A Pathol. Anat. Histopathol. 415, 459-466 N. and Ozawa, H. (1999) The primary calcification 55. Bianco, P., Riminucci, M., Silvestrini, G., Bonucci, in bones follows removal of decorin and fusion of E., Termine, J.D., Fisher, L.W. and Gehron Robey, collagen fibrils. J. Bone Miner. Res. 14, 273-280 P. (1993) Localization of bone sialoprotein (BSP) 67. Prince, C.W., Rahemtulla, E and Butler, W.T. (1984) to Golgi and post-Golgi secretory structures in Incorporation of [35S]sulphate into glycosaminog- osteoblasts and to discrete sites in early bone matrix. lycans by mineralized tissues in vivo. Biochem. J. J. Histochem. Cytochem. 41, 193-203 224, 941-945