Gene Expression and Distribution of Amelogenin during Amelogenesis

1993 Fiscal Year Final Research Report Summary

• Project/Area Number: 04454456
• Research Category: Grant-in-Aid for General Scientific Research (B)
• Allocation Type: Single-year Grants
• Research Field: Morphological basic dentistry
• Research Institution: Nihon University, School of Dentistry
• Principal Investigator: INAGE Toshihiko Nihon University School of Dentistry, Department of anatomy, Associate Professor, 歯学部, 助教授 (90096769)
• Co-Investigator(Kenkyū-buntansha): OHSHIMA Mitsuhiro Nihon University School of Dentistry, Department of anatomy, Associate Professor, 歯学部, 助手 (30194145) ; KUWATA Fumiyuki Nihon University School of Dentistry, Department of Biochemistry. Associate Prof, 歯学部, 講師 (60120440) ; TODA Yoshihisa Nihon University School of Dentistry, Department of anatomy. Professor, 歯学部, 教授 (20059413) ; SHIMOKAWA Hitoyata Tokyo Medical and Dental University, Faculty of Dentistry, Department of Biochem, 歯学部, 助教授 (80014257)
• Project Period (FY): 1992 – 1993
• Keywords: amelogenin / in situ hybridization / immunhistochemistry / mRNA / organ culture / ameloblast / amelogenesis

Research Abstract

Tooth enamel is elaborated through two major processes : matrix formation. At the stage of matrix formation, enamel includes 70 % enamel proteins, and during the maturation stage most of the amelogenin is resorbed. However, little is known about the mechanism of amelogenin resorption. To clarify detail of the mechanism and stages in the secretion and resorption of amelogenin, the gene expression and distribution of amelogenin were studied.
MATERIALS AND METHODS : DDY mice from 13 d in utero to 3 wk after birth were used. The animals were fixed in 4% parafomaldehyde solution then decalciffed in EDTA.Tooth germs of molars and incisors were then embedded in paraffin. [Preparation of RNA probe] cDNA for bovine amelogenin (860 bp) was subcloned in the pSPT18 vector. Then radioactive antisense and sense RNAs were synthesized by RNA poiymerase. In situ hybridization (ISH) was followed by method using Nomura. [Immunohistochemistry (IHC)] A polyclonal bovine antibody against 25-kDa rabbit and 12 C-terminal amino acids was raised and reacted by the ABC method. RESULTS : the signals from the amelogenin mRNA were evident at the inner enamel epithelium. The signals increased in premeloblasts and reached a maximum at the inner and outer enamel secretory regions. From the terminal secretion zone, the signals became gradually weaken toward the incisal edge. Gene expression was observed until the Int. ruffled-ended ameloblasts (RA).IHC using polyclonal antibodies : The reaction was essentially the same as that during the secretory stage. In the maturation zone, the immunoreacivity was obseved in the ruffled border of the int. RA.The immunoreaction for amelogenins was recognized in the 2nd and 3rd RA but the distal ends of the smooth-ended ameloblasts did not show any reaction. IHC using antibody against the C-terminus : The cellular reaction was essentially the same, being evident from the inner enamel epithelium to the initial RA.These results clearly show that amelogenin is synthesized by ameloblasts until the early maturation stage, and then is resorbed from the RA.We also investigated the gene expression of TGF- beta . The gene exoression appeared simultaneously at the same stage with amelogenin, and suggesting that TGF- beta regulates the proliferation, differentiation and synthesis of amelogenin. In cultured molar tooth germs from 18-d fetal mice in utero, enamel was formed after 4 days of culture. TGF- beta 1