Modulation of trophoblast cell death by oxygen and EGF.
BACKGROUND
Preeclampsia, a maternal hypertensive disease, is characterized by shallow invasion of the maternal spiral arterioles resulting in hypoxia/reperfusion type insult; however, the molecular mechanism is unknown. The aim of this study was to determine the mechanism of altered oxygen tension or inhibition of phosphatidyl-inositol-3-kinase (PI3K) on trophoblast survival and to investigate the effect of epidermal growth factor (EGF) on maintaining cellular integrity.


METHODS
We have used flow cytometry, immunoblotting, and fluoroimmunocytochemistry to study apoptosis in a characterized, spontaneously transformed first trimester extravillous-like trophoblast cell line that exhibits many characteristics of in vivo trophoblast.


RESULTS
Time-dependent exposure of first trimester extravillous-like trophoblast to all oxygen tensions tested promoted dissipation of the mitochondrial membrane potential (psi(m)) and resulted in a significant increase in celldeath by 48 hr as determined by dual staining flow cytometry. Western blot analysis revealed expression ofcleaved caspase-3 and caspase-9 increased with time with hypoxia and hyperoxia promoting the greatest elevation indicating that longer duration of exposure to a change inoxygen tension causes increased apoptosis via a mitochondrial-mediated pathway. Disruption of the anti-apoptotic PI3K pathway by LY294002 (40 microM), its specific inhibitor, caused further significant dissipation of the psi(m) (p< 0.01) and cleavage of caspase-3. EGF was able to maintain the psi(m) and to prevent cleavage of caspase-3 even in the presence of LY294002, indicating that its survival effects were independent of the PI3K pathway.


CONCLUSIONS
These results suggest that inhibition of the PI3K/Akt pathway can sensitize first-trimester trophoblast-like cells into oxygen-induced cell death and that EGF exerts its anti-apoptotic effect independently of PI3K/Akt.
Perkins
Jonathan
J
Department of Reproductive and Vascular Biology, The Medical School, University of Birmingham, Birmingham, UK.
St  John
Justin
J
Ahmed
Asif
A
D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D003470:Culture Media; D004815:Epidermal Growth Factor / Q000378:metabolism* / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D010100:Oxygen / Q000378:metabolism* / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D014327:Trophoblasts / Q000378:metabolism*
D003470:Culture Media; D000081082:Phosphoinositide-3 Kinase Inhibitors; D004815:Epidermal Growth Factor; D020169:Caspases; D010100:Oxygen