The ERK-1/2 signaling pathway is involved in the stimulation of branching morphogenesis of fetal mouse submandibular glands by EGF
The ERK-1/2 signaling pathway is involved in the stimulation of branching morphogenesis of fetal mouse submandibular glands by EGF
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DOI：
10.1006/dbio.2000.9639
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发表时间：
2000-04-15
影响因子：
2.7
通讯作者：
Gresik, EW
中科院分区：
文献类型：
--
作者：
Kashimata, M;Sayeed, S;Gresik, EW
我们以前曾报道表皮生长因子（EGF）刺激胚胎小鼠下颌下腺（SMG）的分支形态发生（M。Kashimata和E. W. Gresik，1997，Dev. Dyn. 208，149-161），并且EGF受体（埃格）主要（如果不是唯一的话）位于胎儿SMG的上皮成分上（E. W. Gresik，M. Kashimata，Y.卡多亚河马修斯，N. Minami和S. Yamashina，1997，J. Histochem.细胞化学45，1651-1657）。EGFR是一种受体酪氨酸激酶，在与其配体结合后，它触发几种细胞内信号级联，其中一种激活促分裂原活化蛋白激酶（MAPK）ERK-1/2。在这里，我们研究了EGF是否利用ERK-1/2信号级联刺激胚胎小鼠SMG的分支形态发生。在E14、E16和E18（E0 =阴道栓日）采集SMG雏形作为匹配对;置于成分确定的培养基（BGJb）的威尔斯孔中;暴露于EGF 5或30 min或单独暴露于培养基（对照）。通过Western印迹，我们发现，EGF诱导的含磷酸酪氨酸的蛋白质，包括在170 kDa和44 kDa/42 kDa的条带，大概相应的EGFR和ERK-1/2的磷酸化形式，分别出现多个条带。其他印迹显示磷酸化EGFR和磷酸化ERK-1/2响应EGF的特异性外观。磷酸酪氨酸的免疫组织化学染色增加在质膜EGF刺激后5或30分钟。弥漫性细胞质染色MEK-1/2（MAPK激酶，激活ERK-1/2）增加EGF刺激后的细胞膜附近。磷酸化ERK-1/2被定位在细胞核中的一些上皮细胞EGF后5分钟，但在许多细胞的细胞核中EGF后30分钟。PD 98059，磷酸化和激活的MEK-1/2的抑制剂本身抑制分支形态发生，而且，减少EGF对分支的刺激作用。Western印迹证实，这种抑制剂阻断暴露于EGF的胎儿SMG中ERK-1/2的磷酸化。这些结果表明，ERK-1/2信号级联的组件存在于胎儿SMG的上皮细胞中，它们被EGF激活，并且抑制该级联干扰分支形态发生。然而，EGF并没有引起其他两个MAPK的磷酸化，SAPK/JNK或p38 MAPK，在胎儿SMG。这些结果表明，ERK-1/2信号是负责的，至少部分地，为刺激EGF对胚胎小鼠SMG的分支形态发生的作用。(C)北京大学出版社.
We have previously reported that epidermal growth factor (EGF) stimulates branching morphogenesis of the fetal mouse submandibular gland (SMG) (M. Kashimata and E. W. Gresik, 1997, Dev. Dyn. 208, 149-161) and that the EGF receptor (EGER) is localized principally, if not exclusively, on the epithelial components of the fetal SMG (E. W. Gresik, M. Kashimata, Y. Kadoya, R. Mathews, N. Minami, and S. Yamashina, 1997, J. Histochem. Cytochem. 45, 1651-1657). The EGFR is a receptor tyrosine kinase, and after binding of its ligand, it triggers several intracellular signaling cascades, among them the one activating the mitogen-activated protein kinases (MAPK) ERK-1/2. Here we investigated whether EGF utilizes the ERK-1/2 signaling cascade to stimulate branching morphogenesis in the fetal mouse SMG. SMG rudiments were collected as matched pairs at E14, E16, and E18 (E0 = day of vaginal plug); placed into wells of defined medium (BGJb); and exposed to EGF for 5 or 30 min or to medium alone (controls). By Western blotting we found that EGF induced the appearance of multiple bands of phosphotyrosine-containing proteins, including bands at 170 kDa and 44 kDa/42 kDa, presumably corresponding to the phosphorylated forms of EGFR and ERK-1/2, respectively. Other blots showed the specific appearance of the phosphorylated EGFR and of phospho-ERK-1/2 in response to EGF. Immunohistochemical staining for phosphotyrosine increased at the plasma membrane after EGF stimulation for 5 or 30 min. Diffuse cytoplasmic staining for MEK-1/2 (the MAPK kinase that activates ERK-1/2) increased near the cell membrane after EGF stimulation. Phospho-ERK-1/2 was localized in the nuclei of a few epithelial cells after EGF for 5 min, but in the nuclei of many cells after EGF for 30 min. PD98059, an inhibitor of phosphorylation and activation of MEK-1/2 by itself inhibited branching morphogenesis and, furthermore, decreased the stimulatory effect of EGF on branching. Western blots confirmed that this inhibitor blocked phosphorylation of ERK-1/2 in fetal SMGs exposed to EGF. These results show that components of the ERK-1/2 signaling cascade are present in epithelial cells of the fetal SMG, that they are activated by EGF, and that inhibition of this cascade perturbs branching morphogenesis. However, EGF did not cause phosphorylation of two other MAPKs, SAPK/JNK or p38MAPK, in fetal SMGs. These results imply that the ERK-1/2 signaling is responsible, at least in part, for the stimulatory effect of EGF on branching morphogenesis of the fetal mouse SMG. (C) 2000 Academic Press.