目的

探讨在人骨髓间充质干细胞（hMSC）、小鼠前成骨细胞（MC3T3）和成牙本质细胞（MDPC-23）中，牙本质基质蛋白1（DMP1）是否通过激活丝裂原活化蛋白激酶（MAPK）-细胞外调节蛋白激酶（ERK）信号通路发挥作用。

方法

Western blot 检测不同时间点rDMP1C 和rDMP1F 蛋白处理hMSC、MC3T3-E1 和MDPC-23 后，对MAPK-ERK 的激活情况；并检测使用MAPK 抑制剂后，ERK 的激活是否被抑制；细胞免疫荧光技术观察MAPK 抑制剂抑制激活的ERK 从胞浆向胞核的转位情况。 Western blot 检测siRNA 沉默Ras 基因后，对rDMP1 引起MAPK-ERK 信号通路激活的影响。

结果

三种细胞中，rDMP1F 和rDMP1C 在5 min ～3 h 均可以激活MAPK-ERK 通路，而总ERK 在各时间点均无显著变化；rDMP1F 激活信号通路的持续时间均明显长于rDMP1C；MAPK 抑制剂处理组的p-ERK 条带与rDMP1F/C 处理组的p-ERK 条带差别明显。 hMSC 中，rDMP1F 激活MAPK 信号通路持续时间要长于其在MC3T3 和MDPC-23 中的作用。 细胞免疫荧光检测发现，MAPK 抑制剂组可以阻断ERK 向细胞核内的转位。MC3T3 和MDPC-23 在siRNA 沉默Ras 基因后，ERK 的磷酸化水平与rDMP1F 单独处理组相比显著降低。

结论

rDMP1C 和rDMP1F 都通过Ras 激活MAPK-ERK信号通路，而rDMP1F 比rDMP1C 具有更强的信号功能。

Objective

Dentin matrix phosphoprotein 1 （DMP1） is a non-collagenous， acidic extracellular matrix protein expressed chiefly in bone and dentin. The present study was aimed to investigate whether DMP1 can trigger MAPK signalling pathway in hMSC， MC3T3-E1 and MDPC-23 cells.

Methods

The activation of MAPK-ERK by rDMP1F/C and inhibition of ERK by MAPK inhibitor at different timepoints in hMSC， MC3T3-E1 and MDPC-23 cells was detected using Western blot. The inhibition of ERK cytoplasm/nucleus translocation by the MAPK inhibitor was detected using immunofluorescence microscopy. The activation of MAPK-ERK signalling pathway by DMP1 was further validated through knowdown of Ras gene expression by siRNA.

Results

The MAPK-ERK could be activated by both recombinant DMP1 C-terminal （rDMP1C） and full lenth （rDMP1F） from 5 min to 3 h in three cell lines. The phosphorylated-ERK increased， but not total ERK， in these cell lines treated by rDMP1. The phosphorylation of MAPK-ERK by rDMP1F in hMSC cells last longer than that in MC3T3 or MDPC-23 cells. The activation of MAPK lasted longer by rDMP1F than by rDMP1C. MAPK inhibitor could decrease the phosphorylation of MAPK-ERK by rDMP1C and rDMP1F and inhibit the ERK translocation from the cytoplasm into the nucleus. Knowdown of Ras gene expression by siRNA could significantly decrease the phosphoralated of ERK.

Conclusion

Both rDMP1C and rDMP1F can active the MEK-ERK， however， rDMP1F has a stronger effect in trigger MAPK signalling pathway than rDMP1C.