EGF stimulates proliferation of mouse embryonic stem cells:: involvement of Ca2+ influx and p44/42 MAPKs
EGF stimulates proliferation of mouse embryonic stem cells:: involvement of Ca2+ influx and p44/42 MAPKs
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DOI：
10.1152/ajpcell.00142.2005
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发表时间：
2006-01-01
影响因子：
5.5
通讯作者：
Han, HJ
中科院分区：
文献类型：
--
作者：
Heo, JS;Lee, YJ;Han, HJ
我们研究了 EGF 对小鼠胚胎干 (ES) 细胞增殖及其相关信号通路的影响。 EGF 以时间和剂量依赖性方式增加 [H-3] 胸苷和 5-溴-2'-脱氧尿苷的掺入。 EGF 刺激 EGF 受体 (EGFR) 的磷酸化。用 AG-1478 或除草霉素 A 抑制 EGFR 酪氨酸激酶，用新霉素或 U-73122 抑制 PLC，用双吲哚马来酰亚胺 I 或星形孢菌素抑制 PKC，以及用硝苯地平或甲氧基维拉帕米抑制 L 型 Ca2+ 通道，可防止 EGF 诱导的 [H-3] 胸苷掺入。 PKC-α、-β(I)、-gamma、-delta 和 -zeta 易位至细胞膜，细胞内 Ca2+ 浓度 ([Ca2(+)](i)) 因 EGF 的作用而增加。此外，抑制 EGFR 酪氨酸激酶、PLC 和 PKC 完全阻止了 EGF 诱导的 [Ca2+](i) 增加。 EGF 还可以增加磷酸肌醇的水平，而这种水平可以被 EGFR 酪氨酸激酶抑制剂阻断。此外，EGF 迅速增加 H2O2 的形成，并且用抗氧化剂（N-乙酰基-L-半胱氨酸）预处理抑制 EGF 诱导的 [Ca2+](i) 增加。此外，我们观察到 EGF 引起的 p44/42 MAPK 磷酸化以及 EGFR 酪氨酸激酶、PLC、PKC 或 Ca2+ 通道的抑制可阻断 EGF 诱导的 p44/42 MAPK 磷酸化。用 PD-98059（MEK 抑制剂）抑制 p44/42 MAPK 可减弱 EGF 诱导的 [ 3H] 胸苷掺入增加。最后，抑制 EGFR 酪氨酸激酶、PKC、Ca2+ 通道或 p44/42 MAPK 分别减弱 EGF 刺激的细胞周期蛋白 D1、细胞周期蛋白 E、细胞周期蛋白依赖性激酶 (CDK)2 和 CDK4。总之，EGF 通过 PLC/PKC、Ca2+ 内流和 p44/42 MAPK 信号通路（通过 EGFR 酪氨酸激酶磷酸化）部分刺激小鼠 ES 细胞的增殖。
We examined the effect of EGF on the proliferation of mouse embryonic stem (ES) cells and their related signal pathways. EGF increased [H-3] thymidine and 5-bromo-2'-deoxyuridine incorporation in a time-and dose-dependent manner. EGF stimulated the phosphorylation of EGF receptor ( EGFR). Inhibition of EGFR tyrosine kinase with AG-1478 or herbimycin A, inhibition of PLC with neomycin or U-73122, inhibition of PKC with bisindolylmaleimide I or staurosporine, and inhibition of L-type Ca2+ channels with nifedipine or methoxyverapamil prevented EGF-induced [H-3] thymidine incorporation. PKC-alpha, -beta(I), -gamma, -delta, and -zeta were translocated to the membrane and intracellular Ca2+ concentration ([Ca2(+)](i)) was increased in response to EGF. Moreover, inhibition of EGFR tyrosine kinase, PLC, and PKC completely prevented EGF-induced increases in [Ca2+](i). EGF also increased inositol phosphate levels, which were blocked by EGFR tyrosine kinase inhibitors. Furthermore, EGF rapidly increased formation of H2O2, and pretreatment with antioxidant (N-acetyl-L-cysteine) inhibited EGF-induced increase of [Ca2+](i). In addition, we observed that p44/42 MAPK phosphorylation by EGF and inhibition of EGFR tyrosine kinase, PLC, PKC, or Ca2+ channels blocked EGF-induced phosphorylation of p44/42 MAPKs. Inhibition of p44/42 MAPKs with PD-98059 (MEK inhibitor) attenuated EGF-induced increase of [ 3H] thymidine incorporation. Finally, inhibition of EGFR tyrosine kinase, PKC, Ca2+ channels, or p44/42 MAPKs attenuated EGF-stimulated cyclin D1, cyclin E, cyclin-dependent kinase (CDK)2, and CDK4, respectively. In conclusion, EGF partially stimulates proliferation of mouse ES cells via PLC/PKC, Ca2+ influx, and p44/42 MAPK signal pathways through EGFR tyrosine kinase phosphorylation.