The molecular biological activity of PTH has been exclusively assigned to the aminoterminal [1-34] region, leaving no activity or biological role for the carboxyl-terminal portion. However, binding studies using biologically active, intact radiolabelled PTH have demonstrated the existence of separate binding sites for both the aminoterminal and the carboxyl-terminal regions. Whether it is involved in directing the action or the actual binding to the receptor, the role of the carboxyl-binding site remains unexplained.

PTH affects the mineralization of dentine in vivo as well as the odontogenesis of molar explants in vitro. This suggests that PTH may play an important part in the mineralization of enamel and dentine. Cultivation of the tooth germ in the bell stage is well known to be a good experimental system for studying the differentiation of the tooth-forming cells as well as their matrix secretion. It also provides a useful tool for investigating both the direct action of drugs on tooth formation and the mechanisms of drug action.

Our preliminary examination showed notable effects of four PTH fragments [1-34, 13-34, 39-68, 53-84] on alkaline phosphatase activity in a mandibular first molar dissected from a 1-day-old mouse. Treatment of the tooth germ with PTH[1-34] for 24 hr significantly decreased alkaline phosphatase activity; PTH[53-84], on the other hand, significantly increased it. Other fragments did not influence this enzyme activity. This indicated that alkaline phosphatase activity in the tooth germ could be influenced by the carboxyl-terminal as well as the aminoterminal region of PTH.

Our aim now was to compare in more detail the effect of carboxyl-terminal fragment PTH[53-84] with that of aminoterminal fragment PTH[1-34] on tooth germ development, to gain a better understanding of the physiological significance of the carboxyl-terminal. For this purpose, the effects of these PTH fragments on odontogenesis at various developmental stages were evaluated by measuring alkaline phosphatase activity, which has been related to the formation and mineralization of hard tissues, and by examining the histological features of cultured tooth germs.

Time-pregnant ddY mice, purchased from Japan SLC, Inc. (Hamamatu, Japan), were caged in plastic tubs covered with stainless-steel tops and containing hardwood-chip bedding, under automatically controlled conditions of temperature (23±1°C), humidity (50±10%), and a 12-hr light:dark cycle, and were fed a standard laboratory diet ad libitum (CE2; Crea Co., Tokyo, Japan). The first appearance of the vaginal plug was designated as day 0. Several days later, tooth buds were dissected from the 16-, 17-, 18- and 19-day-old embryos. The lower jaws were then removed, and the first mandibular molars dissected asceptically in Eagle’s minimum essential medium (Gibco Oriental, Tokyo, Japan). The explant was supported by a piece of membrane filter (Nuclepore; Nomura Micro Science Co. Ltd, Tokyo, Japan) on a metal grid and cultured by Trowell-type organ culture. The medium consisted of BGJb medium, supplemented with 10% (V/V) fetal bovine serum (Cell Culture Laboratories, Cleveland, OH, U.S.A.), 100 IU/ml penicillin, and 100 ?g/ml streptomycin. The culture dishes were kept in a humidified incubator at 37°C in an atmosphere of 5% CO₂ in air. The explants were cultured for 2 or 7 days in the absence or presence of human PTH[1-34] (0.1 ?M) or human PTH[53-84] (0.1 ?M) (Peptide Institute, Osaka, Japan). At the end of culturing, the explants were sonicated for 1 min before the determination of alkaline phosphatase activity. Alkaline phosphatase activity was assayed by determining the release of p-nitrophenol from p-nitrophenylphosphate, according to the method of Lowry (1957). Other explants were fixed with 10% neutral buffered formalin, embedded in paraffin and sectioned serially at 4?m. The sections were stained with haematoxylin and eosin, and evaluated by light microscopy. Statistical differences between the control and experimental groups were analysed by Student’s t-test.

Table 1(A) shows the effects of PTH[53-84] and PTH[1-34] on alkaline phosphatase activity in the tooth germs at different developmental stages. The enzyme activity increased significantly with PTH[1-34] (0.1 ?M) at day 16 (cap stage) and day 17 (early bell stage), but it decreased at day-18 (advanced bell stage) and day 19 (late bell stage). On the other hand, alkaline phosphatase activity was significantly lower by treatment with PTH[53-84] (0.1 ?M) at day 16 but higher at day 19 than in the controls. This effect was also observed during cultivation of day-17 embryonic mouse molar tooth germs for 7 days, during which they developed from the early bell stage to mineralization of dentine. In the first three of the 7 days of cultivation, the mesenchymal cells in the early bell stage differentiated into odontoblasts and secreted predentine. In the last 3 days the tooth germs developed from the advanced to the late bell stage. When tooth germs were treated with PTH[1-34] (0.1 ?M) for the first 3 days, alkaline phosphatase activity increased, as shown in Table 1(B). Also, tooth germs treated with PTH[53-84] (0.1 ?M) for the last 3 days had significantly increased alkaline phosphatase activity. On the other hand, inhibitory effects on alkaline phosphatase activity were observed in tooth germs treated with PTH[1-34] for the last 3 days and PTH[53-84] for the first 3 days.

Fig. 1. Light micrographs showing the in vitro effects of PTH[1-34] and PTH[53-84] on tooth germs. Day-17 embryonic mouse tooth germs cultured in the absence (A, B) or presence of PTH[1-34] (C, D) and PTH[53-84] (E, F). Sections stained with haematoxylin and eosin, x250. (A) Mesial cusp of tooth germ cultured with control medium for 3 days (ob; odontoblasts, pd; predentine). (B) Mesial cusp of tooth germ cultured with control medium for 7 days. Note differentiation of ameloblasts and odontoblasts and formation of dentine (d; dentine, ab; ameloblasts, e; enamel). (C) Tooth germs cultured with PTH[1-34] (0.1 ?M) for the first 3 days. Note formation of enamel matrix. (D) Tooth germs cultured with PTH[1-34] (0.1 ?M) for the last 3 days. Note vacuolated ameloblasts. (E) Tooth germs cultured with PTH[53-84] (0.1 ?M) for the first 3 days. Note absence of enamel and a small quantity of dentine. (F) Tooth germ cultured with PTH[53-84] (0.1 ?M) for the last 3 days. Note formation of enamel matrix.

The effects of the PTH fragments on alkaline phosphatase activity were accompanied by histological changes in the mesial cusp of the first molar. In tooth germs cultured for 7 days in control medium, the mesenchymal cells had differentiated into odontoblasts and deposited a small amount of dentine. The inner enamel epithelium had already differentiated into pre-ameloblasts with proximally located nuclei and enamel was partially formed. As shown in Fig. 1(C, F), significant differences were observed histologically in the mesial cusp of the first molar tooth germ treated with PTH[1-34] (0.1 ?M) for the first 3 days and PTH[53-84] (0.1 ?M) for the last 3 days. Both treatments were associated with increased formation of enamel and dentine. These effects might be dependent upon the development stage of the germ. On the other hand, as shown in Fig. 1 (D,E), tooth germs treated with PTH[1-34] (0.1 ?M) for the last 3 days and PTH[53-84] (0.1 ?M) for the first 3 days showed a slight decrease of dentine and enamel matrix, in spite of the presence of ameloblasts, odontoblasts and stratum intermedium. A number of vacuolated ameloblasts and vesiculation of the cells were also observed.

A recent study has demonstrated that the interaction of the aminoterminal and carboxyl-terminal regions of a PTH molecule with their own respective binding sites on the cells seems to occur in a fairly independent manner. Here, we show, to the best of our knowledge for the first time, that carboxyl-terminal human PTH[53-84] as well as aminoterminal human PTH[1-34] have marked effects on both alkaline phosphatase activity and histological features in mouse tooth germs in culture. These effects of both fragments depended on the developmental stage and were opposite at the same developmental stage. During the cultivation of day-17 mouse molar tooth germs for 7 days, the early, advanced and late bell stages are successively seen as a gradient starting from the cusp tips and proceeding cervically. At the early developmental stage, odontogenesis as evaluated by alkaline phosphatase activity and histological features was stimulated by PTH[1-34] but suppressed by PTH[53-84]. On the other hand, at the late developmental stage, it was stimulated by PTH[53-84] but suppressed by PTH[1-34]. The findings clearly show that not only the aminoterminal but also the carboxyl-terminal fragment of PTH has a distinct effect on each developmental stage in the tooth germs, which observation is supported by the observed biological effects of carboxyl-terminal PTH fragments on osteoblasts, osteoclasts, and neonatal condylar cartilage.