Amelogenin (AMELX), the predominant matrix protein in enamel formation, contains a singular phosphorylation site at Serine 16 (S16) that greatly enhances AMELX's capacity to stabilize amorphous calcium phosphate (ACP) and inhibit its transformation to apatitic enamel crystals. To explore the potential role of AMELX phosphorylation in vivo, we developed a knock-in (KI) mouse model in which AMELX phosphorylation is prevented by substituting S16 with Ala (A). As anticipated, AMELX KI mice displayed a severe phenotype characterized by weak hypoplastic enamel, absence of enamel rods, extensive ectopic calcifications, a greater rate of ACP transformation to apatitic crystals, and progressive cell pathology in enamel-forming cells (ameloblasts). In the present investigation, our focus was on understanding the mechanisms of action of phosphorylated AMELX in amelogenesis. We have hypothesized that the absence of AMELX phosphorylation would result in a loss of controlled mineralization during the secretory stage of amelogenesis, leading to an enhanced rate of enamel mineralization that causes enamel acidification due to excessive proton release. To test these hypotheses, we employed microcomputed tomography (µCT), colorimetric pH assessment, and Fourier Transform Infrared (FTIR) microspectroscopy of apical portions of mandibular incisors from 8-week old wildtype (WT) and KI mice. As hypothesized, µCT analyses demonstrated significantly higher rates of enamel mineral densification in KI mice during the secretory stage compared to the WT. Despite a greater rate of enamel densification, maximal KI enamel thickness increased at a significantly lower rate than that of the WT during the secretory stage of amelogenesis, reaching a thickness in mid-maturation that is approximately half that of the WT. pH assessments revealed a lower pH in secretory enamel in KI compared to WT mice, as hypothesized. FTIR findings further demonstrated that KI enamel is comprised of significantly greater amounts of acid phosphate compared to the WT, consistent with our pH assessments. Furthermore, FTIR microspectroscopy indicated a significantly higher mineral-to-organic ratio in KI enamel, as supported by µCT findings. Collectively, our current findings demonstrate that phosphorylated AMELX plays crucial mechanistic roles in regulating the rate of enamel mineral formation, and in maintaining physico-chemical homeostasis and the enamel growth pattern during early stages of amelogenesis.

中文翻译：

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釉原蛋白磷酸化在调节牙釉质形成中的作用


Amelogenin (AMELX) 是牙釉质形成中的主要基质蛋白，在丝氨酸 16 (S16) 处含有一个单一的磷酸化位点，可大大增强 AMELX 稳定无定形磷酸钙 (ACP) 并抑制其转化为磷灰石牙釉质晶体的能力。为了探索 AMELX 磷酸化在体内的潜在作用，我们开发了一种敲入 (KI) 小鼠模型，其中通过用 Ala (A) 取代 S16 来阻止 AMELX 磷酸化。正如预期的那样，AMELX KI 小鼠表现出严重的表型，其特征是牙釉质发育不全、牙釉质棒缺失、广泛异位钙化、ACP 转化为磷灰石晶体的比率较高以及牙釉质形成细胞（成釉细胞）中进行性细胞病理学。在本研究中，我们的重点是了解磷酸化 AMELX 在釉质形成中的作用机制。我们假设，缺乏 AMELX 磷酸化会导致牙釉质生成分泌阶段受控矿化的丧失，从而导致牙釉质矿化速率加快，从而由于过多的质子释放而导致牙釉质酸化。为了检验这些假设，我们对 8 周龄野生型 (WT) 和 KI 小鼠的下颌切牙根尖部分采用显微计算机断层扫描 (μCT)、比色 pH 评估和傅里叶变换红外 (FTIR) 显微光谱分析。正如假设的那样，μCT 分析表明，与 WT 相比，KI 小鼠在分泌阶段的牙釉质矿物质致密化率显着更高。 尽管牙釉质致密化速度更快，但在釉质生成的分泌阶段，最大 KI 牙釉质厚度的增加速度明显低于 WT，在成熟中期达到的厚度约为 WT 的一半。 pH 值评估显示，与 WT 小鼠相比，KI 分泌釉质的 pH 值较低，正如假设的那样。 FTIR 结果进一步表明，与 WT 相比，KI 牙釉质的酸性磷酸盐含量明显更高，这与我们的 pH 值评估一致。此外，FTIR 显微光谱表明 KI 牙釉质中矿物质与有机物的比率显着较高，μCT 结果也支持这一点。总的来说，我们目前的研究结果表明，磷酸化 AMELX 在调节牙釉质矿物质形成速率、维持牙釉质形成早期阶段的物理化学稳态和牙釉质生长模式方面发挥着至关重要的机制作用。