Torres-Quintana M A, Lécolle S, Septier D, Palmier B, Rani S, MacDougall M, Goldberg M
Laboratoire de Biologie et Physiopathologie Craniofaciales-Groupe Matrices Extrcellulaires et Biominéralisations, Faculté de Chirurgie Dentaire, Université René Descartes-Paris V, France.
J Dent Res. 2000 Oct;79(10):1794-801. doi: 10.1177/00220345000790101101.
Post-translational modification of enamel proteins is regulated by casein kinases (CK) and results in binding sites for calcium ions that subsequently play a key role during the initial stages of mineralization. Phosphorylation may also influence the secretion and extracellular organization of enamel proteins. Previous studies indicated that inositol hexasulphate inhibited the activity of CK-I and/or CK-II in mouse tooth germs (Torres-Quintana et al., 1998). We hypothesized that inositol hexasulphate would also inhibit the activity of the specific casein kinase(s) identified in secretory ameloblasts, and would prove useful for determination of the extent to which phosphorylation might influence the organization of enamel proteins at early stages of enamel formation. To test this hypothesis, we dissected mandibular first molars from 18-day-old mouse embryos and cultured them for 11 days in the presence of 0-0.1 mM inositol hexasulphate. Ultastructural analysis revealed that the formation of enamel was largely impaired at an inhibitor concentration > or = 0.08 mM. Quantitative radioautographic analysis of [33P]phosphate incorporation indicated that radiolabeled phosphate normally secreted into forming enamel was retained within ameloblasts. In contrast, no significant difference was observed between control and inositol-hexasulphate-treated tooth germs when cultures were labeled with [3H]serine and [3H]proline. SDS-PAGE and Western blot analysis confirmed that while inositol hexasulphate inhibited CK-mediated phosphorylation, it did not significantly alter protein synthesis. We conclude that impairment of phosphorylation leads to intracellular accumulation of [3H]phosphate-containing material by ameloblasts. We also conclude that when non-phosphorylated enamel matrix proteins are secreted, they are either unable to form an enamel matrix that supports mineralization, or they diffuse throughout a poorly mineralized dentin.
釉质蛋白的翻译后修饰受酪蛋白激酶（CK）调控，其结果是产生钙离子结合位点，这些位点在矿化初期发挥关键作用。磷酸化还可能影响釉质蛋白的分泌和细胞外组织。先前的研究表明，肌醇六硫酸盐可抑制小鼠牙胚中CK-I和/或CK-II的活性（Torres-Quintana等人，1998年）。我们推测，肌醇六硫酸盐也会抑制分泌性成釉细胞中特定酪蛋白激酶的活性，并将证明其有助于确定磷酸化在釉质形成早期对釉质蛋白组织的影响程度。为了验证这一假设，我们从18日龄小鼠胚胎中分离出下颌第一磨牙，并在含有0 - 0.1 mM肌醇六硫酸盐的条件下培养11天。超微结构分析显示，当抑制剂浓度≥0.08 mM时，釉质的形成受到很大损害。对[33P]磷酸盐掺入的定量放射自显影分析表明，正常分泌到正在形成的釉质中的放射性标记磷酸盐保留在成釉细胞内。相比之下，当用[3H]丝氨酸和[3H]脯氨酸标记培养物时，在对照牙胚和经肌醇六硫酸盐处理的牙胚之间未观察到显著差异。SDS-PAGE和蛋白质印迹分析证实，虽然肌醇六硫酸盐抑制了CK介导的磷酸化，但并未显著改变蛋白质合成。我们得出结论，磷酸化受损导致成釉细胞内积累含[3H]磷酸盐的物质。我们还得出结论，当分泌未磷酸化的釉质基质蛋白时，它们要么无法形成支持矿化的釉质基质，要么扩散到矿化不良的牙本质中。
