Department of Orthodontics, Applied Life Sciences, Hiroshima University, Institute of Biomedical & Health Sciences, Kasumi, Minami-ku, Hiroshima, Japan.
J Periodontol. 2018 Apr;89(4):496-505. doi: 10.1002/JPER.17-0087.
Amelogenins are a family of enamel matrix proteins that are important for formation of enamel. Amelogenins may induce division of mesenchymal stem cells (MSCs), among others. Recently, the C-terminus of the amelogenin peptide (AMG-CP) has been shown to enhance the proliferation of cementoblast lineage cells. The role of the amelogenin peptide on the proliferation of human MSCs and related alterations in the intracellular signaling pathway were studied.
MSCs were exposed to AMG-CP in vitro. The MTS and 5-bromo-2'-deoxyuridine (BrdU) assays were used to determine proliferation. Expression of the amelogenin receptor, lysosomal-associated membrane protein 1 (LAMP1), was examined in MSCs with western blotting. Binding of AMG-CP to LAMP1 was inhibited with anti-LAMP1 antibody. Components of the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) pathway were studied with western blotting and enzyme-linked immunosorbent assay, and U0126, an MAPK inhibitor, was used to inhibit ERK activity.
MSC proliferation was significantly increased in the presence of AMG-CP and significantly inhibited by anti-LAMP1 antibody or U0126. Increased phosphorylated ERK1/2 was observed in the presence of AMG-CP, and decreased phosphorylated ERK1/2 was seen in the presence of anti-LAMP1 antibody or U0126.
A C-terminal amelogenin variant increased the proliferation of MSCs via an interaction with LAMP1 and the MAPK-ERK signaling pathway, indicating the possibility of using MSCs for tissue regeneration in the craniofacial region.
釉原蛋白是一组牙釉质基质蛋白，对于牙釉质的形成很重要。釉原蛋白可能会诱导间充质干细胞（MSCs）等细胞的分裂。最近，釉原蛋白肽的 C 端（AMG-CP）已被证明可以增强成牙骨质细胞系细胞的增殖。本研究旨在探讨釉原蛋白肽对人 MSCs 增殖的作用及其对细胞内信号通路的相关改变。
体外将 MSCs 暴露于 AMG-CP 中。使用 MTS 和 5-溴-2'-脱氧尿苷（BrdU）检测法来确定增殖情况。通过 Western blot 检测 MSCs 中釉原蛋白受体溶酶体相关膜蛋白 1（LAMP1）的表达。用抗 LAMP1 抗体抑制 AMG-CP 与 LAMP1 的结合。通过 Western blot 和酶联免疫吸附试验研究丝裂原活化蛋白激酶（MAPK）-细胞外信号调节激酶（ERK）通路的组成部分，并使用 MAPK 抑制剂 U0126 抑制 ERK 活性。
AMG-CP 存在时，MSC 增殖明显增加，而抗 LAMP1 抗体或 U0126 则明显抑制增殖。在 AMG-CP 存在的情况下观察到磷酸化 ERK1/2 增加，而在存在抗 LAMP1 抗体或 U0126 的情况下观察到磷酸化 ERK1/2 减少。
C 端釉原蛋白变体通过与 LAMP1 和 MAPK-ERK 信号通路相互作用增加 MSCs 的增殖，这表明有可能利用 MSCs 进行颅面区域的组织再生。
