Laboratory of Molecular Signaling and Stem Cells Therapy, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing 100050, China.
Cell Prolif. 2013 Aug;46(4):447-56. doi: 10.1111/cpr.12039. Epub 2013 Jul 6.
Mesenchymal stem cells (MSCs) are a reliable resource for tissue regeneration, but their molecular mechanisms of differentiation and proliferation remain unclear; this situation has restricted use of MSCs to a limited number of applications. A previous study of ours found a member of the epidermal growth factor family, epiregulin (EREG), to be involved in regulation of MSC differentiation. In the present study, we have used human dental stem cells from the apical papilla (SCAPs) to investigate the role of EREG on proliferation of MSCs.
SCAPs were isolated from apical papillae of immature third molars. Retroviral short hairpin RNA (shRNA) was used to silence EREG gene expression, and human recombinant EREG protein was used to stimulate SCAPs. SCAP proliferation was examined using tetrazolium dye colorimetric assay/cell growth curve. Western blotting was performed to detect expressions of extracellular signal-regulated protein kinases 1 and 2 (Erk1/2), mitogen-activated protein kinases 1 and 2 (MEK1/2), protein kinase B (Akt), p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK).
Depletion of EREG with shRNA inhibited SCAP proliferation and repressed phosphorylation of Erk1/2 and JNK. Human recombinant EREG protein promoted cell proliferation and enhanced Erk1/2, MEK and JNK phosphorylation in SCAPs. Furthermore, blocking MEK/Erk signalling with specific Erk1/2 inhibitor PD98059, or JNK signalling with specific inhibitor SP600125, abolished effects of EREG on cell proliferation.
These findings indicate that EREG could enhance cell proliferation in dental tissue-derived MSCs by activating MEK/Erk and JNK signalling pathways.
间充质干细胞（MSCs）是组织再生的可靠资源，但它们的分化和增殖的分子机制尚不清楚；这种情况限制了 MSCs 在少数应用中的使用。我们之前的一项研究发现表皮生长因子家族的一个成员，表皮调节素（EREG），参与调节 MSC 的分化。在本研究中，我们使用来自根尖乳头的人牙髓干细胞（SCAPs）来研究 EREG 对 MSC 增殖的作用。
SCAPs 从未成熟第三磨牙的根尖乳头中分离出来。逆转录病毒短发夹 RNA（shRNA）用于沉默 EREG 基因表达，人重组 EREG 蛋白用于刺激 SCAPs。使用四唑染料比色法/细胞生长曲线检测 SCAP 增殖。Western blot 用于检测细胞外信号调节蛋白激酶 1 和 2（Erk1/2）、丝裂原活化蛋白激酶 1 和 2（MEK1/2）、蛋白激酶 B（Akt）、p38 丝裂原活化蛋白激酶（p38 MAPK）和 c-Jun N-末端激酶（JNK）的表达。
用 shRNA 耗尽 EREG 抑制了 SCAP 的增殖，并抑制了 Erk1/2 和 JNK 的磷酸化。人重组 EREG 蛋白促进细胞增殖，并增强了 SCAPs 中 Erk1/2、MEK 和 JNK 的磷酸化。此外，用特定的 Erk1/2 抑制剂 PD98059 或 JNK 抑制剂 SP600125 阻断 MEK/Erk 信号通路，消除了 EREG 对细胞增殖的影响。
这些发现表明，EREG 通过激活 MEK/Erk 和 JNK 信号通路，可增强牙组织来源的 MSC 中的细胞增殖。
