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 osteogenic activity on osteoblast-like cells (ST2) and human periodontal ligament (HPDL) cells. Results: Fraction 3 (Fr.3) and a transforming growth factor- beta 1 (TGF-b1) control reduced alkaline phosphatase (ALP) activity in ST2 but enhanced ALP activity in HPDL cells. The enhanced ALP activity was blocked by anti-TGF-b antibodies. Furthermore, using a dual-luciferase reporter assay, we dem- onstrated that Fr.3 can induce the promoter activity of the plasminogen activator inhibitor type 1 (PAI-1) gene. Conclusion: These results show that the osteoinductive ac- tivity of enamel extracts on HPDL cells is mediated by TGF-b1. J Periodontol 2006;77:1688-1694. KEY WORDS Periodontal ligament; transforming growth factor beta1. P roteins extracted from the imma- ture enamel matrix of developing teeth possess important biologic activities, such as the induction of osteo- genesis 1-3 and cementogenesis. 4 For example, it was shown in in vivo and in vitro systems that enamel matrix deriv- atives (EMDs) have cementum- and osteopromotive activities 5 and stimulate the proliferation and differentiation of osteoblastic cells. 6 In developing dental enamel, there are five major extracellular matrix proteins: three structural proteins and two protein- ases. The cDNAs encoding these proteins have been cloned from the developing tooth germs of various mammals, such as bovines, humans, pigs, rats, and mice. The three structural proteins are amelo- genin, 7 enamelin, 8 and sheathlin, 9,10 also known as ameloblastin 11 or amelin. 12 The two proteinases are enamelysin 13,14 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 formation 1 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 assumed that the major structural compo- nents of the developing enamel matrix are responsible for the observed osteoinductive * Department of Periodontics and Endodontics, School of Dental Medicine, Tsurumi University, Yokohama, Japan. † Department of Biochemistry, School of Dental Medicine, Tsurumi University. ‡ Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, MI. § Department of Periodontology, Nihon University School of Dentistry at Matsudo, Chiba, Japan. doi: 10.1902/jop.2006.050352 Volume 77 • Number 10 1688