179 Journal of Oral Science, Vol. 43, No. 3, 179-188, 2001 Gene expression and immunohistochemical localization of decorin and biglycan in association with early bone 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 osteoblasts before the onset of matrix small proteoglycans, decorin and biglycan, which are mineralization and that they could play a role in the associated with osteoblast differentiation, and how this earliest stages of bone formation. Negative proteoglycan 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 gene 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 proteins Immunohistochemistry showed that newly differenti- such as proteoglycans, osteocalcin (bone Gla protein, ated osteoblasts expressed biglycan moderately, decorin BGP), matrix Gla protein (MGP), osteonectin, osteopontin weakly, and osteocalcin and BSP faintly. The (OPN), bone sialoprotein (BSP), and bone acidic unmineralized bone matrices among the osteoblasts glycoprotein-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 glycosaminoglycan 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 dermatan sulfate 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 glycoproteins 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.