Department of Oral and Craniofacial Sciences, University of Pittsburgh School of Dental Medicine, 335 Sutherland Drive (UPSDM), Pittsburgh, PA 15260, USA; Center for Craniofacial Regeneration, UPSDM, Pittsburgh, PA, USA.
Department of Endodontics, UPSDM, Pittsburgh, PA, USA.
Matrix Biol. 2024 Aug;131:17-29. doi: 10.1016/j.matbio.2024.05.004. Epub 2024 May 16.
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.
成釉蛋白（AMELX）是釉质形成中的主要基质蛋白，其丝氨酸 16 位（S16）存在单一的磷酸化位点，极大地增强了 AMELX 稳定无定形磷酸钙（ACP）和抑制其转化为磷灰石牙晶体的能力。为了探索 AMELX 磷酸化在体内的潜在作用，我们构建了一个敲入（KI）小鼠模型，其中 S16 被丙氨酸（A）取代，从而阻止 AMELX 磷酸化。正如预期的那样，AMELX KI 小鼠表现出严重的表型特征，表现为釉质发育不全、缺乏釉柱、广泛的异位钙化、ACP 向磷灰石晶体转化的速度加快，以及釉质形成细胞（成釉细胞）的进行性细胞病理学。在本研究中，我们的重点是了解磷酸化 AMELX 在釉质发生中的作用机制。我们假设 AMELX 磷酸化的缺失会导致釉质发生分泌期的矿化失控，导致釉质矿化速度加快，由于过多质子的释放，导致釉质酸化。为了验证这些假设，我们采用 microcomputed tomography（µCT）、比色 pH 评估和下颌切牙根尖的傅里叶变换红外（FTIR）微光谱分析来自 8 周龄野生型（WT）和 KI 小鼠。正如假设的那样，µCT 分析表明，在分泌期，KI 小鼠的釉质矿化密度明显高于 WT。尽管釉质矿化速度加快，但在釉质发生的分泌期，KI 的最大釉质厚度增加速度明显低于 WT，达到中期成熟时的厚度约为 WT 的一半。如假设的那样，pH 评估显示 KI 小鼠的分泌釉质中的 pH 较低。FTIR 结果进一步表明，KI 釉质中含有明显更多的酸性磷酸盐，与我们的 pH 评估结果一致。此外，FTIR 微光谱分析表明，KI 釉质的矿物质/有机物比值明显高于 WT，这与 µCT 结果一致。总之，我们目前的研究结果表明，磷酸化 AMELX 在调节釉质矿化形成速度以及在釉质发生早期维持物理化学平衡和釉质生长模式方面发挥着重要的机制作用。
