Epidermal growth factor abrogates hypoxia-induced apoptosis in cultured human trophoblasts through phosphorylation of BAD Serine 112.
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.
Humphrey
Rachel G
RG
Department of Obstetrics-Gynecology, Washington University School of Medicine, 4566 Scott Avenue, St. Louis, Missouri 63110, USA.
Sonnenberg-Hirche
Christina
C
Smith
Steven D
SD
Hu
Chaobin
C
Barton
Aaron
A
Sadovsky
Yoel
Y
Nelson
D Michael
DM
D017209:Apoptosis / Q000187:drug effects*; D015687:Cell Hypoxia / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology*; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D011247:Pregnancy; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D014327:Trophoblasts / Q000187:drug effects* / Q000378:metabolism; D051025:bcl-Associated Death Protein / Q000235:genetics / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D051025:bcl-Associated Death Protein; D049452:Green Fluorescent Proteins; D012694:Serine; D004815:Epidermal Growth Factor; D048051:p38 Mitogen-Activated Protein Kinases