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Noggin Blocks Osteoinductive Activity of Porcine Enamel Extracts

¹ Section of Periodontology, Department of Hard Tissue Engineering, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan; and
² Department of Biochemistry, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama, Kanagawa 230-8501, Japan;

*corresponding author, takanori.peri{at}tmd.ac.jp

	ABSTRACT

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Enamel extracts induce biomineralization such as osteogenesis and cementogenesis, but the molecular component responsible for this activity remains uncertain. We fractionated enamel extracts from developing pig teeth and isolated the osteoinductive fraction. Proteins from pig enamel scrapings were extracted under alkaline conditions (pH 10.8) and fractionated with the use of a Sephadex G-100 (size exclusion) column. The ability of each fraction to enhance alkaline phosphatase (ALP) activity was assayed in ST2 cells, a mouse bone marrow stromal cell line. The osteoinductive fraction of enamel extracts (OFE) was found in fractions 44 and 45, which induced ST2 cells to express the phenotype of bone-forming osteoblasts, and to form mineralized nodules. Furthermore, the ALP activity of ST2 cells exposed to OFE was reduced by noggin, an antagonist of BMPs, and OFE reacted with BMP-2/4 antibody in dot-blot analysis. These results indicate that OFE contains BMPs that contribute to the induction of biomineralization.

KEY WORDS: enamel extracts • bone morphogenetic proteins (BMPs) • noggin.

	INTRODUCTION

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Enamel extracts have bioactivity, such as the induction of osteogenesis (Urist, 1971) and cementogenesis (Hammarström, 1997). In addition, EMDOGAIN^®, a mixture of porcine enamel extracts, has osteopromotive activity in vivo (Boyan et al., 2000) and stimulates the proliferation and differentiation of osteoblastic cell lines in vitro (Schwartz et al., 2000). However, the mechanisms involved in these processes are not well-understood.

Three matrix proteins, corresponding to amelogenin (Hu et al., 1996), enamelin (Hu et al., 1997b), and sheathlin (Hu et al., 1997a), and two enzymes, corresponding to MMP-20 (Fukae et al., 1998) and EMSP1 (Simmer et al., 1998), have been purified and the cDNAs cloned from developing porcine teeth. However, whether these purified enamel proteins are themselves related to bone formation or periodontal regeneration remains unknown.

Some members of the bone morphogenetic protein (BMP) family, which constitutes a part of the transforming growth factor-beta (TGF-beta) superfamily (Kingsley, 1994), can induce osteogenesis in vivo (Urist, 1965) and osteogenic differentiation in vitro (Yamaguchi et al., 1996). During tooth development, ameloblasts express enamel proteins as well as mRNA for BMP-2, BMP-4, BMP-5, and BMP-7 (Åberg et al., 1997), and BMP-7 has been detected in immature enamel by immunochemistry (Helder et al., 1998). The present study isolates an osteoinductive fraction from enamel extracts (OFE) and investigates whether its osteoinductive activity depends on BMPs.

	MATERIALS & METHODS

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Extraction and Fractionation of the Enamel Proteins
Tooth germs of permanent molars were dissected from fresh mandibles of approximately six-month-old pigs purchased from a slaughterhouse. The animal experimental protocol used in this study is in agreement with the standards of the Tokyo Medical and Dental University. Surrounding soft and pulpal tissues were removed, and the surface of immature enamel was rinsed in cold saline, wiped with Kimwipes, and scraped (Fukae and Shimizu, 1974). Pooled enamel samples were suspended in 10 volumes of 0.05 M Sörensen buffer (pH 7.4) and homogenized by means of a Polytron homogenizer (Kinematica, Littau, Switzerland) for 30 sec at half speed. The homogenate was centrifuged for 10 min at 10,000 x g. This procedure was repeated three times. The supernatants were combined as the neutral soluble fraction. The pellets were suspended in 10 volumes of 0.05 M carbonate-bicarbonate buffer (pH 10.8), and extracted in the same manner as the neutral buffer extraction. The carbonate buffer supernatants were collected as the alkaline soluble fraction. The pellets were dissolved with 0.5 M acetic acid and collected as the acid soluble fraction (Tanabe et al., 1992).

Gel Filtration
The alkaline soluble fraction was applied to a column of Sephadex G-100 (4 x 100 cm) equilibrated with 0.05 M carbonate-bicarbonate buffer (pH 10.8) and run at a flow rate of 15 mL/hr. The eluate was monitored at 280 nm and collected in 5-mL fractions. An aliquot of each fraction was de-salted on a PD-10 column (Amersham-Pharmacia Biotech, Uppsala, Sweden) in 0.5 M acetic acid and lyophilized.

Acrylamide Gel Electrophoresis
Samples were resolved by 15% polyacrylamide slab gels containing 1% SDS (SDS-PAGE) as described by Laemmli (1970) and stained with Coomassie Brilliant Blue R-250.

Cell Culture
The mouse bone marrow stromal cell line ST2 (Riken Cell Bank, Tsukuba, Japan) was cultured in the alpha modification of Eagle's medium (MEM; Life Technologies, Grand Island, NY, USA) containing 10% fetal bovine serum (Asahi Technoglass, Chiba, Japan) and 1% antibiotics [100 U/mL of penicillin-G and 100 µg/mL of streptomycin sulfate (Gibco BRL, Grand Island, NY, USA)] at 37°C in a humidified 5% CO₂ atmosphere.

Enzyme Assay
ST2 cells were spread on 96-well plates at a density of approximately 5 x 10⁴ cells/well and incubated for 24 hrs. Growth medium was then changed to contain 200 nM all-trans retinoic acid (Sigma, St. Louis, MO, USA) and G-100 fractions dissolved in dH₂O. After 72 additional hrs of incubation, the cells were washed once with PBS, and ALP activity was determined with 10 mM p-nitrophenylphosphate as the substrate in 100 mM 2-amino-2-methyl-1, 3-propanediol-HCl buffer (pH 10.0) containing 5 mM MgCl₂ and incubated for 8 min at 37°C. The addition of NaOH quenched the reaction, and the absorbance at 405 nm was read on a plate reader (Bio-Rad Model 450, Hercules, CA, USA).

Mitogenic Assay
The mitogenic activity of the osteoinductive fraction of enamel extracts (OFE) was assayed by means of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) (Sigma, St. Louis, MO, USA) according to the manufacturer's instructions. ST2 cells were incubated in 100 µL of growth medium at an initial density of 1.6 x 10³ cells/well. After 24 hrs, the medium was changed to MEM with 1.0% FBS and incubated for another 24 hrs. The medium was then changed to contain concentrations of OFE ranging from 0 to 10 µg/mL. After 24 hrs, 10 µL of MTT (5 mg/mL) was added to each well, and the cells were incubated for 4 hrs. The medium was then discarded, and 100 mL of dimethylsulfoxide was added to each well. The absorbance of each well was measured at 570 nm with background subtraction at 655 nm in a microplate reader.

Differentiation Assay
ST2 cells were plated in 24-well plates at an initial density of 9.0 x 10³ cells/well. After 24 hrs, the medium was replaced with growth medium containing 50 µM ascorbic acid, 10 mM ß-glycerophosphate, and 200 nM retinoic acid (differentiation medium) and OFE concentrations ranging from 0 to 50 µg/mL. The medium was changed every 72 hrs.

Alizarin Red Staining
A solution of 1% alizarin red S (sodium alizarin sulfonate, Sigma, St. Louis, MO, USA) in dH₂O was adjusted to pH 6.4 with 0.1 N ammonium hydroxide. The cells were fixed in 100% methanol, stained with alizarin red S for 10 min, then washed with dH₂O and photographed. The stained area was quantified by NIH Image (NIH, Bethesda, MD, USA).

Polymerase Chain Reaction (PCR)
Total RNA was extracted by the use of acid guanidinium thiocyanate-phenol-chloroform (Chomczynski and Sacchi, 1987) from ST2 cells that were cultured in the differentiation medium with or without 50 µg/mL of OFE for 1, 4, 7, 11, or 14 days. Thereafter, cDNA was synthesized from 3 µg of the total RNA by means of an oligo-dT primer and the You-prime First-Strand Beads kit (Amersham-Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's protocol. PCR primers were designed based upon mouse mRNA sequences in the GenBank database. The primer pairs were as follows: ß-actin, 5'-GACGATATCGCTGCGATGGT and 5' -ATCTTTTCACGGTTGGCCT (353 bp amplimer); osteocalcin, 5' -TCTGACAAAGCAAGCAGGAG and 5' -AAATAGTGATA CCGTAGATGCG (191 bp); bone sialoprotein (BSP), 5' -ACCGGCCACGCTACTTTCTTT and 5' -GACCGCCAGCTCG TTTTCA (431 bp); ALP, 5'-TCTTCTTGCTGGTGGAAGGAG and 5'-GGAGACATTTTCCCGTTCACC (317 bp). The PCR reactions proceeded as follows: 5 min of denaturation at 94°C, followed by specific numbers of cycles consisting of denaturation at 94°C for 30 sec, primer annealing at 55°C for 30 sec, and elongation at 72°C for 30 sec. A final elongation proceeded at 72°C for 5 min. The PCR products were resolved by 4.5% polyacrylamide gel electrophoresis and stained with ethidium bromide.

Noggin Binding Assay
ST2 cells were seeded in 96-well plates at a density of approximately 5 x 10⁴ cells/well and incubated for 24 hrs. Before the medium was changed, various concentrations of noggin (recombinant mouse noggin/Fc chimera, Genzyme-Techne, Minneapolis, MN, USA) and OFE (40 µg/mL) were bound at room temperature for 1 hr in the growth medium (Zimmerman et al., 1996). Seventy-two hours after the medium was changed, ALP activity was determined as described above in "Enzyme Assay".

Dot-blot Immunoassay
Samples were blotted onto PVDF membranes (Bio-Rad, Hercules, CA, USA) that had been soaked with PBS. The blotted membrane was rinsed with PBS and incubated in 0.5% skim milk in PBS containing 0.05% Tween 20 for 30 min, to block non-specific binding. The membrane was then incubated for 1 hr at room temperature with biotinylated anti-human BMP-2/4 (1:500 dilution, R&D systems, Minneapolis, MN, USA) in PBS containing 0.05% Tween 20. The membrane was washed and immunostained with avidin-biotin complex (Hsu and Raine, 1981).

Statistical Analysis
All values are represented as means ± standard error (SE). Statistical significance was determined by an unpaired Student's t test, and p < 0.05 was considered statistically significant.

	RESULTS

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Isolation of Osteoinductive Fraction from Porcine Enamel Extracts (OFE)
Approximately 13.3 g (wet weight) of immature enamel was obtained from unerupted porcine molars. Approximately 1.0 g (dry weight) of total protein was extracted, as determined by the method of Bradford (1976), with BSA as the standard. The alkaline soluble fraction induced the osteoblastic differentiation of ST2 cells, whereas the neutral and acid fractions did not (data not shown). We then fractionated the alkaline soluble fraction.

Porcine enamel proteins were separated into five fractions (Fig. 1A). The SDS-PAGE profiles corresponding to I, II, III, IV, and V are shown in Fig. 1B. The elution of almost all enamel proteins on this system depended on their molecular size, except the first eluted peak that contained low-molecular-weight proteins (Fig. 1B). We considered that the proteins in the first fraction formed an aggregate (Tanabe et al., 1992). Fig. 1A shows that the activity was contained within fractions 36 to 50. We selected fractions 44 and 45 as the "osteoinductive fraction of enamel extracts (OFE)". The SDS-PAGE profile of fractions 36 to 50 shows that OFE contains mainly 25-, 23-, and 20-kDa proteins, all of which are considered as amelogenins (Yamakoshi et al., 1994) (Fig. 1C). These three isoforms were abundant in fractions 48, 49, and 50, respectively.

View larger version (59K): [in this window] [in a new window]   Figure 1. Elution profile of porcine enamel extracts from Sephadex G-100 column and ALP activity of ST2 cells induced by each fraction. (A) The chromatogram represents absorbance at 280 nm. Bars show ALP activities of ST2 cells exposed to G-100 fractions. Data are means ± SE of three culture wells. SDS-PAGE profiles of fractions of I to V (B) and G-100 fractions from No. 36 to No. 50 (C) stained with CBB. Molecular weights (Bio-Rad Low Range Standards, Hercules, CA, USA) are shown in left margin.

The RT-PCR results showed that osteocalcin expression increased at days 11 and 14 in the presence of OFE, and that BSP increased in the presence of OFE at each experimental period (Fig. 2A). At the end of the experimental period (day 15), cultures incubated in the differentiation medium were stained with alizarin red S (Fig. 2B). OFE increased the size of the stained area at concentrations ranging from 12.5 to 50 µg/mL in a dose-dependent manner.

View larger version (38K): [in this window] [in a new window]   Figure 2. (A) Effect of 50 µg/mL OFE on osteocalcin, bone sialoprotein (BSP), and alkaline phosphatase (ALP) mRNA expression in cultures of ST2 cells. Total RNA of ST2 cells incubated with or without OFE was collected on days 1, 4, 7, 11, and 14. Each culture was incubated with 50 µM ascorbic acid, 10 mM ß-glycerophosphate, and 200 nM retinoic acid. (B) Nodule formation induced by OFE. OFE was added to medium at concentrations of 0, 12.5, 25, and 50 µg/mL. Nodule cultures were stained with 2% alizarin red S on day 15. The stained area was quantified by NIH Imaging. Data are means ± SE of three culture wells. Significantly different from culture without OFE at *p < 0.05, **p < 0.001.

View larger version (17K): [in this window] [in a new window]   Figure 3. The influence of noggin incubated with OFE on ALP activity of ST2 cells. OFE (40 µg/mL) and noggin (1, 10, 100, or 300 ng/mL) were incubated for 1 hr and then added to the cells. ALP activity was measured 72 hrs later. Data are means ± SE of three culture wells. Significantly different from culture without noggin at *p < 0.05.

View larger version (57K): [in this window] [in a new window]   Figure 4. Dot-blot analysis of BMP2/4 protein in OFE. Lane 1, 50 µg of OFE; lane 2, 500 ng of rhBMP-2 (Genzyme-Techne, USA; positive control); and lane 3, 50 µg of BSA (Sigma, St. Louis, MO, USA; negative control). Each sample was diluted as shown in left margin.

	DISCUSSION

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ST2 is a well-characterized bone marrow stromal cell line that does not exhibit typical features of the osteoblast phenotype in control cultures (Ogawa et al., 1988). After gel filtration of the alkaline soluble fraction by Sephadex G-100, the osteoinductive fraction of enamel extracts (OFE) enhanced ALP activity in ST2 cells and induced them to express an osteoblast-like phenotype, such as up-regulated osteocalcin and BSP mRNA expression, and the formation of mineralized nodules in the differentiation medium. We also confirmed the up-regulations in the expression of mRNA of ALP, osteocalcin, and BSP within 24 hrs when cells were cultured in the medium which did not contain ascorbic acid or ß-glycerophosphate.

It was reported that low-molecular-mass amelogenin from bovine dentin had chondrogenic activity (Nebgen et al., 1999), and recombinant rat low-molecular-mass amelogenins induced signaling effects in vivo and in vitro (Veis et al., 2000). In this study, OFE was considered to contain mainly 25-, 23-, and 20-kDa amelogenins. However, the fractions 48, 49, and 50 corresponding to the peak consisting of 25-, 23-, and 20-kDa amelogenin, respectively, did not stimulate ALP activity in ST2 cells (Fig. 1A). Besides 25-, 23-, and 20-kDa amelogenins, the fractions around OFE contain many aggregates, as reported previously (Tanabe et al., 1992). Therefore, OFE might contain low-molecular-mass amelogenins which have osteogenic activity.

Demineralized enamel extracts as well as bone extracts have osteoinductive ability (Urist, 1971). These active proteins, which have been purified from bovine bone and cloned as BMPs (Wang et al., 1988; Wozney et al., 1988), are members of the TGF-beta superfamily (Kingsley, 1994). BMPs are known sources of osteoinduction in vivo and in vitro. BMP-2 and BMP-4 induce osteocalcin production and stimulate ALP activity in ST2 cells (Yamaguchi et al., 1996). The present study confirmed the up-regulation of osteocalcin and BSP mRNA expression.

During tooth development, BMP-2, BMP-4, BMP-5, and BMP-7 mRNAs are expressed in mouse ameloblasts (Åberg et al., 1997) that cover immature enamel and secrete enamel proteins. BMP-7 has been detected in immature enamel by immunochemistry (Helder et al., 1998). Therefore, BMPs are secreted from ameloblasts and should be present in enamel extracts. This study shows that the osteogenic activity of enamel extracts can be attributed to BMPs.

A growing number of secreted proteins have been discovered that antagonize BMPs functions. These include noggin, chordin, follistatin, and the DAN family (Miyazono, 2000). These BMP antagonists share the functional property of binding specifically to BMPs, thus preventing interaction with their receptors. Noggin, which is one of these antagonists, binds to BMP-2, BMP-4, and GDF-6 with high affinity, but to BMP-7 with low affinity (Zimmerman et al., 1996; Chang and Hemmati-Brivanlou, 1999). The present study found that noggin reduced ALP activity of ST2 cells exposed to OFE. This indicated that OFE contains BMPs that were antagonized by noggin. We performed a gel shift assay to ensure that noggin had no interaction with any amelogenins in OFE. We could not detect any specific shifted bands (data not shown). In addition, our dot-blot immunoassay showed that OFE reacted against human BMP-2/4 antibody. We also tried Western blot analyses; however, we could not detect any bands corresponding to BMPs. These results might be caused by the denaturing of protein. Then we performed RT-PCR to confirm the expressions of BMP mRNAs and BMP receptor mRNAs in ameloblasts. Using specific primer sets based upon mouse and human mRNA sequences, we detected the mRNAs of BMP2, BMP4, BMP5, BMP7, BMP receptor IA, and BMP receptor IB in the cDNA library of porcine secretory ameloblasts, as previously described (Oida et al., 2002; data not shown).

This is the first study to show that porcine enamel extracts contain BMPs. Since the concentration is very low, the detection of BMPs in enamel extracts is difficult. However, we successfully identified BMP activity in enamel extracts by using the ST2 cell culture system, which is highly sensitive to these proteins. BMPs in enamel extracts could explain the observed induction of biomineralization by enamel matrix proteins.

	ACKNOWLEDGMENTS

 
We thank all the members of our labs, especially Dr. Y. Yamakoshi, Dr. T. Karakida, and Dr. T. Nagano. This work was partially supported by a grant-in-aid (No. 12671818), funding from the Bio-Venture and High Technology Research Centers Project from the Japanese Ministry of Education, Culture, Sports, Science and Technology. Y. Morotome was a recipient of Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists.

Received October 24, 2001; Last revision March 19, 2002; Accepted April 4, 2002

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