Torres-Quintana M A, Lécolle S, Goldberg M
Laboratoire de Biologie et Physiopathologie Crânio-faciales, Université René Descartes, Paris V, Faculté de Chirurgie Dentaire, Montrouge, France.
Arch Oral Biol. 1998 Aug;43(8):597-610. doi: 10.1016/s0003-9969(98)00037-5.
To study the effects of impaired protein phosphorylation on dentine formation and mineralization, inositol hexasulphate, an intracellular type I and type II casein kinase inhibitor, was used in an in vitro organotypic culture system. Mandibular first molar tooth germs were dissected from 18-day-old mouse embryos and cultured for 11 days with and without inositol hexasulphate at different concentrations. At 0.04-0.08 mM inhibitor, cellular alterations were not detected. Dentine displayed the characteristic purple-blue colour when Stains all, a specific stain for extracellular phosphoproteins, was used. At 0.1 mM, dentine failed to stain and mineralization did not occur, as seen from the von Kossa method. The presence of numerous lysosome-like vesicles inside cells indicated that the experiment was at the limits of cytotoxicity; higher concentrations induced severe cellular alterations. Therefore, quantitative radioautography was carried out on germs treated or not with the inhibitor at 0.1 mM. [33P]-phosphate incorporation showed that grain density in inhibited germs compared with that in control germs was about double in odontoblasts and half in the predentine/dentine compartment. In the presence of inositol hexasulphate the incorporation of [3H]serine into odontoblast cell bodies was unchanged between 2 and 24 h while in predentine/dentine, grain density was higher between 1 and 4 h, and reduced at 24 h. Both with [33P]phosphate and [3H]serine, labelling was seen throughout the porous dentine formed in vitro and not as a band located at the predentine/dentine junction, as is the case in vivo. With [3H]proline, in the presence of the inhibitor, a small reduction of grain density occurred in cell bodies, no significant difference was seen between 1 and 4 h in predentine/dentine, and more silver grains were present after 24 h both in cells and in the matrix. The radioautographic data support the view that the inhibitor interacts mostly with post-transductional phosphorylation and does not alter significantly other cell synthetic pathways and functions. Finally, the experiments presented here confirm that phophorylated proteins have a key role in dentine mineralization.
为研究蛋白质磷酸化受损对牙本质形成和矿化的影响，在体外器官型培养系统中使用了肌醇六硫酸盐，一种细胞内I型和II型酪蛋白激酶抑制剂。从18日龄小鼠胚胎中分离出下颌第一磨牙牙胚，在有和没有不同浓度肌醇六硫酸盐的情况下培养11天。在0.04 - 0.08 mM抑制剂浓度下，未检测到细胞改变。当使用细胞外磷蛋白的特异性染色剂“全染剂”时，牙本质呈现出特征性的紫蓝色。在0.1 mM时，从冯·科萨法可见牙本质未能染色且未发生矿化。细胞内存在大量溶酶体样小泡表明该实验处于细胞毒性的极限；更高浓度会诱导严重的细胞改变。因此，对用0.1 mM抑制剂处理或未处理的牙胚进行了定量放射自显影。[33P] - 磷酸盐掺入显示，与对照牙胚相比，受抑制牙胚中，成牙本质细胞中的颗粒密度约为两倍，而前期牙本质/牙本质区域中的颗粒密度为一半。在存在肌醇六硫酸盐的情况下，[3H]丝氨酸掺入成牙本质细胞体在2至24小时之间没有变化，而在前期牙本质/牙本质中，颗粒密度在1至4小时之间较高，在24小时时降低。无论是[33P]磷酸盐还是[3H]丝氨酸，标记都出现在体外形成的多孔牙本质中，而不是像在体内那样位于前期牙本质/牙本质交界处的一条带中。对于[3H]脯氨酸，在存在抑制剂的情况下，细胞体中的颗粒密度略有降低，前期牙本质/牙本质在1至4小时之间没有显著差异，24小时后细胞和基质中都有更多的银颗粒。放射自显影数据支持这样的观点，即抑制剂主要与转导后磷酸化相互作用，并且不会显著改变其他细胞合成途径和功能。最后，这里展示的实验证实磷酸化蛋白在牙本质矿化中起关键作用。
