Epidermal growth factor abrogates hypoxia-induced apoptosis in cultured human trophoblasts through phosphorylation of BAD serine 112
Epidermal growth factor abrogates hypoxia-induced apoptosis in cultured human trophoblasts through phosphorylation of BAD serine 112
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DOI：
10.1210/en.2007-1253
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发表时间：
2008-05-01
期刊：
影响因子：
4.8
通讯作者：
Nelson, D. Michael
中科院分区：
文献类型：
--
作者：
Humphrey, Rachel G.;Sonnenberg-Hirche, Christina;Nelson, D. Michael
我们测试的假设，表皮生长因子（EGF）限制缺氧诱导的细胞凋亡在培养的人滋养层细胞的凋亡蛋白Bcl-2相关的死亡启动子（BAD）的磷酸化。从妊娠38-40周的无并发症妊娠的胎盘中分离细胞滋养层细胞。原代滋养层细胞或转染的JEG 3滋养层细胞在低于1%或20%的氧气中在EGF和信号传导途径抑制剂存在或不存在下培养。使用免疫印迹法定量BAD、绿色荧光蛋白（GFP）-BAD、14-3-3、Bcl-X-L和在细胞角蛋白18中间丝的凋亡切割期间形成的新表位。通过共聚焦显微镜分析荧光抗体免疫染色的培养物的BAD和GFP。荧光共振能量转移法检测内源性BAD与GFP-BAD之间的相互作用。我们发现EGF在标准培养条件下增加了BADser 112的磷酸化。而缺氧增强细胞凋亡和增加磷酸化的BADser 136和BADser 155，缺氧减少磷酸化的BADser 112，这种效果是可逆的EGF。转染的GFP-BAD通过共定位和荧光共振能量转移与内源性BAD相互作用，增强了缺氧诱导的JEG 3细胞凋亡。EGF减少缺氧JEG 3细胞中的细胞凋亡，过度表达GFP-BAD，但不过度表达GFP-BAD的细胞中，在112位点具有丝氨酸到丙氨酸的突变。免疫共沉淀研究表明，EGF减少了BAD与Bcl-X-L的促凋亡相互作用。EGF对BADser 112磷酸化的影响依赖于p38 MAPK的作用。我们的结论是EGF信号通过p38 MAPK增加BADser 112的磷酸化，从而限制滋养层细胞凋亡。
We tested the hypothesis that epidermal growth factor (EGF) limits hypoxia-induced apoptosis in cultured human trophoblasts by phosphorylation of the proapoptotic protein Bcl-2-associated death promoter (BAD). Cytotrophoblasts were isolated from placentas of uncomplicated pregnancies at 38-40 wk gestation. Primary trophoblasts or transfected JEG3 trophoblast cells were cultured in less than 1 or 20% oxygen in the presence or absence of EGF and signaling pathway inhibitors. BAD, green fluorescent protein (GFP)-BAD, 14-3-3, Bcl-X-L, and neoepitopes formed during apoptotic cleavage of cytokeratin 18 intermediate filaments were quantified using immunoblotting. Cultures immunostained by fluorescent antibodies were analyzed by confocal microscopy for BAD and GFP. Fluorescence resonance energy transfer was used to detect molecular interaction between endogenous BAD and GFP-BAD. We found EGF increased the phosphorylation of BADser112 under standard culture conditions. Whereas hypoxia enhanced apoptosis and increased phosphorylation of both BADser136 and BADser155, hypoxia diminished phosphorylation of BADser112, and this effect was reversible by EGF. Transfected GFP-BAD, which directly interacted with endogenous BAD by colocalization and fluorescence resonance energy transfer, enhanced hypoxia-induced apoptosis in JEG3 cells. EGF reduced apoptosis in hypoxic JEG3 cells that overexpressed GFP-BAD but not in cells overexpressing GFP-BAD that harbored a serine-to-alanine mutation at the 112 site. Coimmunoprecipitation studies showed that EGF reduced the proapoptotic interaction of BAD with Bcl-X-L. The effect of EGF on phosphorylation of BADser112 was dependent on the action of p38 MAPK. We conclude that EGF signals via p38 MAPK to increase phosphorylation of BADser112 and thereby limit trophoblast apoptosis.