Department of Orthodontics and Craniofacial Developmental Biology, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8553, Japan.
Cell Tissue Res. 2010 Nov;342(2):205-12. doi: 10.1007/s00441-010-1064-7. Epub 2010 Oct 22.
Amelogenins are enamel matrix proteins that play a crucial role in enamel formation. Recent studies have revealed that amelogenins also have cell signaling properties. Although amelogenins had been described as specific products of ameloblasts, recent research has demonstrated their expression in bone marrow stromal cells. In this study, we examined the effect of recombinant human full-length amelogenin (rh174) on the proliferation of human mesenchymal stem cells (MSCs) derived from bone marrow and characterized the associated changes in intracellular signaling pathways. MSCs were treated with rh174 ranging in dose from 0 to 1,000 ng/ml. Cell proliferative activity was analyzed by bromodeoxyuridine (BrdU) immunoassay. The expression of lysosomal-associated membrane protein 1 (LAMP1), a possible amelogenin receptor, in MSCs was analyzed. Anti-LAMP1 antibody was used to block the binding of rh174 to LAMP1. The MAPK-ERK pathway was examined by Cellular Activation of Signaling ELISA (CASE) kit and western blot analysis. A specific MAPK inhibitor, U0126, was used to block ERK activity. It was shown that rh174 increased the proliferation of MSCs and MAPK-ERK activity. The MSC proliferation and MAPK-ERK activity enhanced by rh174 were reduced by the addition of anti-LAMP1 antibody. Additionally, the increased proliferation of MSCs induced by rh174 was inhibited in the presence of U0126. In conclusion, it is demonstrated that rh174 increases the proliferation of MSCs by interaction with LAMP1 through the MAPK-ERK signaling pathway, indicating the possibility of MSC application to tissue regeneration in the orofacial region.
釉原蛋白是牙釉质基质蛋白，在牙釉质形成中发挥着关键作用。最近的研究表明，釉原蛋白还具有细胞信号转导特性。尽管釉原蛋白曾被描述为成釉细胞的特异性产物，但最近的研究表明其在骨髓基质细胞中表达。在这项研究中，我们研究了重组人全长釉原蛋白（rh174）对骨髓来源的人间充质干细胞（MSCs）增殖的影响，并对相关的细胞内信号通路变化进行了特征分析。用 0 至 1000ng/ml 剂量的 rh174 处理 MSCs。通过溴脱氧尿苷（BrdU）免疫测定分析细胞增殖活性。分析 MSCs 中溶酶体相关膜蛋白 1（LAMP1）的表达，LAMP1 可能是釉原蛋白的受体。用抗 LAMP1 抗体阻断 rh174 与 LAMP1 的结合。通过细胞信号转导 ELISA（CASE）试剂盒和 Western blot 分析检测 MAPK-ERK 通路。用特定的 MAPK 抑制剂 U0126 阻断 ERK 活性。结果表明 rh174 增加了 MSCs 的增殖和 MAPK-ERK 活性。添加抗 LAMP1 抗体可降低 rh174 增强的 MSC 增殖和 MAPK-ERK 活性。此外，在 U0126 存在的情况下，rh174 诱导的 MSCs 增殖增加被抑制。结论：rh174 通过与 LAMP1 的相互作用，通过 MAPK-ERK 信号通路增加 MSCs 的增殖，表明 MSC 应用于口腔颌面部组织再生的可能性。
