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Porcine Enamel Protein Fractions Contain Transforming Growth

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Porcine Enamel Protein Fractions Contain Transforming Growth Volume 77 • Number 10 Porcine Enamel Protein Fractions Contain Transforming Growth Factor-b1 Takatoshi Nagano,* Shinichiro Oida,† Shinichi Suzuki,* Takanori Iwata,‡ Yasuo Yamakoshi,‡ Yorimasa Ogata,§ Kazuhiro Gomi,* Takashi Arai,* and Makoto Fukae† Background: Enamel extracts are biologically active and capable of inducing osteogenesis and cementogenesis, but the specific molecules carrying these activities have not been as- certained. The purpose of this study was to identify osteogenic factors in porcine enamel extracts. Methods: Enamel proteins were separated by size-exclusion chromatography into four fractions, which were tested for their roteins extracted from the imma- osteogenic activity on osteoblast-like cells (ST2) and human ture enamel matrix of developing periodontal ligament (HPDL) cells. Pteeth possess important biologic Results: Fraction 3 (Fr.3) and a transforming growth factor- activities, such as the induction of osteo- beta 1 (TGF-b1) control reduced alkaline phosphatase (ALP) genesis1-3 and cementogenesis.4 For activity in ST2 but enhanced ALP activity in HPDL cells. The example, it was shown in in vivo and in enhanced ALP activity was blocked by anti-TGF-b antibodies. vitro systems that enamel matrix deriv- Furthermore, using a dual-luciferase reporter assay, we dem- atives (EMDs) have cementum- and onstrated that Fr.3 can induce the promoter activity of the osteopromotive activities5 and stimulate plasminogen activator inhibitor type 1 (PAI-1) gene. the proliferation and differentiation of Conclusion: These results show that the osteoinductive ac- osteoblastic cells.6 tivity of enamel extracts on HPDL cells is mediated by TGF-b1. In developing dental enamel, there are J Periodontol 2006;77:1688-1694. five major extracellular matrix proteins: three structural proteins and two protein- KEY WORDS ases. The cDNAs encoding these proteins Periodontal ligament; transforming growth factor beta1. have been cloned from the developing tooth germs of various mammals, such as bovines, humans, pigs, rats, and mice. * Department of Periodontics and Endodontics, School of Dental Medicine, Tsurumi University, Yokohama, Japan. The three structural proteins are amelo- † Department of Biochemistry, School of Dental Medicine, Tsurumi University. genin,7 enamelin,8 and sheathlin,9,10 also ‡ Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, 11 12 Ann Arbor, MI. known as ameloblastin or amelin. § Department of Periodontology, Nihon University School of Dentistry at Matsudo, Chiba, The two proteinases are enamelysin13,14 Japan. and kallikrein-4 (KLK4), which is also known as enamel matrix serine protein- ase 1 (EMSP-1).15 Ameloblastin and amelin, first cloned from rat-tooth spe- cific cDNA libraries, are homologs of porcine sheathlin. The periodontal regeneration activity of EMD is generally accepted. This activ- ity stimulates bone formation1 or peri- odontal regeneration,4,16 but the specific molecules that mediate these activities and their mechanisms of action are not well understood. Although it is generally assumedthatthemajorstructuralcompo- nents of the developingenamel matrix are responsiblefortheobservedosteoinductive doi: 10.1902/jop.2006.050352 1688 J Periodontol • October 2006 Nagano, Oida, Suzuki, et al. properties, recently, the induction of alkaline phos- orthodontic reasons. Informed consent was obtained phatase (ALP) activity in ST2 cells by fractionated por- from all patients under a protocol approved by the cine enamel extracts was largely blocked by noggin, a Ethics Committee of Tsurumi University. Human peri- bone morphogenetic protein (BMP) inhibitor.1 Further- odontal ligament (HPDL) cells were obtained as pre- more, EMDi and transforming growth factor-beta viously described by Somerman et al.25 We selected (TGF-b) stimulate rapid translocation of Smad 2 into one-cell type HPDL cells, which had a positive and the nucleus of oral epithelial and fibroblastic cells.17 stable ALP activity. These HPDL cells were used to Some BMPs, which belong to the TGF-b superfam- determine osteoblastic differentiation. The cells were ily,18 can induce osteogenesis in vivo19 and osteo- maintained in the a modification of Eagle’s medium genic differentiation in vitro.20 Thus, some evidence (a-MEM)†† containing 10% fetal bovine serum suggests that growth factors in developing dental (FBS)‡‡ and 1% antibiotics (100 U/ml penicillin-G enamel may mediate the tissue regeneration of EMDs. and 100 mg/ml streptomycin sulfate§§)at37°Cina Thepurposeofthisstudywastoinvestigateandidentify humidified 5% CO2 atmosphere. The osteoblast-like the bioactive agents in fractionated enamel proteins. cell ST2,ii a mouse bone marrow stromal cell line, was cultured in the same condition. MATERIALS AND METHODS Mitogenic Assay All experimental procedures involving the use of ani- The mitogenic activity of fractionated enamel protein mals were reviewed and approved by the Institutional samples were assayed using 3-(4,5)-dimethyl-2-thia- Animal Care Program at Tsurumi University. zolyl-2,5-diphenyl-2H tetrazolium bromide (MTT)¶¶ Extraction and Fractionation of Enamel Proteins according to the manufacturer’s instructions. The Tooth germs of permanent molars were dissected HPDL cells were incubated in 100 ml growth medium from the fresh mandibles of 6-month-old pigs pur- at an initial density of 3 · 103 cells/well. After 24 hours chased from a slaughterhouse. Soft tissues were re- of incubation, the a-MEM containing 1.0% FBS and 50 moved, and the mineralized portion of the developing mg/ml of samples was then changed. After 96 hours, teeth was rinsed in cold saline and wiped.¶ Secretory 10 ml MTT (5mg/ml) was added to each well, and the stage enamel was obtained by scraping the enamel cells were incubated for 4 hours. The medium was dis- surface with a curet.21 Pooled enamel scrapings were carded, and 100 ml dimethylsulfoxide was added to suspended in 10 volumes of 0.05 M So¨rensen buffer each well. The absorbance of each well was measured (pH 7.4) and homogenized by means of a homoge- at 570 nm with background subtraction at 655 nm nizer# for 30 seconds at half speed. The homogenate using a microplate reader.## was centrifuged for 10 minutes at 10,000 · g. This Enzyme Assay (ALP activity) procedure was repeated three times. The superna- The HPDL cells were distributed in 96-well plates at a tants were combined as the neutral soluble fraction. density of approximately 5 · 105 cells/well and incu- The pellets were suspended in 10 volumes of 0.05 M bated for 24 hours. The growth medium was changed carbonate-bicarbonate buffer (pH 10.8) and ex- to contain with or without 10 nM 1a,25-dihydroxyvita- tracted in the same manner as with the neutral buffer. min D *** and 50 mg/ml enamel protein fractions dis- The carbonate buffer supernatants were collected as 3 solved in ultrapure water. After 96 additional hours the alkaline soluble fraction.22,23 of incubation, the cells were washed once with phos- Gel Filtration Chromatography phate buffered saline (PBS), and ALP activity was as- The alkaline soluble fraction was applied to a column sayed using 10 mM p-nitrophenylphosphate as the of sephadex G-100 (4 · 100 cm) equilibrated with substrate in 100 mM 2-amino-2-methyl-1,3-pro- 0.05 M carbonate-bicarbonate buffer (pH 10.8) and panediol-HCl buffer (pH 10.0) containing 5 mM MgCl2 run at a flow rate of 15 ml/hour. The eluate was mon- and incubated for 10 minutes at 37°C. Adding NaOH itored at 280 nm and collected in four fractions. An quenched the reaction, and the absorbance at 405 nm aliquot of each fraction was de-salted on a PD-10 was read on a plate reader. column** in 0.5 M acetic acid and lyophilized. Acrylamide Gel Electrophoresis Samples were resolved by 15% polyacrylamide slab i Emdogain, Biora, Malmo¨, Sweden. ¶ Kimwipes, Kimberly-Clark, Roswell, GA. gels containing 1% sodium dodecyl sulfate (SDS) as # Polytron, Kinematica, Littau, Switzerland. described by Laemmli24 and stained with Coomassie ** Amersham-Pharmacia Biotech, Uppsala, Sweden. †† Life Technologies, Grand Island, NY. brilliant blue (CBB) R-250. ‡‡ Asahi Technoglass, Chiba, Japan. §§ Gibco BRL, Grand Island, NY. Cell Culture ii Riken Cell Bank, Tsukuba, Japan. ¶¶ Sigma, St. Louis, MO. Three periodontally healthy premolars were collected ## Bio-Rad Model 450, Bio-Rad, Hercules, CA. from three patients who had undergone extraction for *** Calbiochem, La Jolla, CA. 1689 Enamel Matrix Contains TGF-b1 Volume 77 • Number 10 The ST2 cells were spread on 96-well plates The compartments ofcellswere fixed in 100% meth- at a density of 1 · 106 cells/well and incubated for anol, stained with alizarin red S for 10 minutes, washed 24 hours. The growth medium was changed to contain with ultrapure water, and photographed to examine with or without 100 nM all-trans retinoic acid††† and the biomineralization activity. The staining solution 50 mg/ml fractionated enamel protein samples dis- was prepared to be 1% alizarin red S (sodium alizarin solved in ultrapure water. After 96 additional hours sulfonate)†††† dissolved in ultrapure water and ad- of incubation, the ALP activity was determined as de- justed to pH 6.4 with 0.1 N ammonium hydroxide. scribed above. These samples and exogenous growth For measuring the calcium contents, the compart- factors (BMP 2:500 ng/ml; TGF-b 1:50 ng/ml) were ments of cells were dissolved by 0.5 N hydrochloric tested for ALP activity. acid. The resulting solution was measured using a test- ing kit and following the manufacturer’s protocol.‡‡‡‡ Anti-TGF-b Antibody Binding Assay The absorbance at 570 nm was read on a plate reader. HPDL cells were distributed in 96-well plates at a density of ;5 · 105 cells/well, and the ST2 cells Statistical Analysis were spread on 96-well plates at a density of 1 · 106 All values are represented as means – SE. Statistical cells/well and incubated for 24 hours. Before the me- significance was determined using an unpaired Stu- dium was changed, various concentrations of anti- dent t test, and P <0.01 was considered statistically TGF-b antibody‡‡‡ and Fraction 3 (Fr.3; 30 mg/ml) significant.
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