Regulation of placenta growth factor by NFkappaB: Potential implications for preeclampsia

Preeclampsia is one of the leading causes of maternal and fetal morbidity and mortality in the world. It is characterized clinically by maternal hypertension and proteinuria after 20 weeks of gestation. In normal pregnancies, levels of Placenta Growth Factor (PlGF) in maternal serum increase dramatically around 15 weeks of gestation and remain high until about 31 weeks gestation when they decline. However, during preeclampsia, this increase in circulating PlGF, produced primarily by trophoblast cells in the placenta, does not occur. This is accompanied by an increase in soluble VEGF Receptor-1 (sFlt-1) production, further limiting bioavailability of PlGF. It is hypothesized that this imbalance of angiogenic factors contributes to pathology seen in preeclampsia. Regulatory factors that contribute to trophoblast PlGF expression are not well known. Our goal was to investigate if active NFkappaB could regulate PlGF expression in trophoblast and to elucidate a mechanism of action for PlGF regulation. Hypoxia has been shown to decrease trophoblast production of PlGF in vitro and transcription factors GCM-1 and MTF-1 have been shown to be positive regulators of PlGF transcription in trophoblast. Little else is known about the molecular mechanisms which regulate PlGF expression in trophoblast. Placental hypoxia and elevated maternal proinflammatory cytokine levels are believe to occur in preeclamptic women, both of which can activate the NFkappaB pathway. However, effects of the NFkappaB pathway on PlGF expression in trophoblast have not previously been investigated. To begin, overexpression vectors of NFkappaB protein p65 and p50 were transiently transfected into JEG-3 cells with a reporter cassette driven by 1.5kb, 828bp and 698bp fragments of the human PlGF promoter. Overexpression of p65 in particular was able to strongly downregulate PlGF transcriptional activity of various lengths of the PlGF promoter. In contrast, p50 displayed less robust and more variable results than p65. Endogenous PlGF mRNA was also significantly decreased in trophoblast with p65 overexpression when measured by quantitative RT-PCR. However, PlGF mRNA expression was increased 2 fold in non-trophoblast hEK293 cells. Overexpression of p65 significantly inhibits GCM-1 transcriptional activity as well as mRNA expression in trophoblast suggesting NFkappaB may downregulate PlGF expression by modulating GCM-1 activity. Finally, we also show hypoxia is capable of activating the canonical NFkappaB activity in trophoblast. Attempts to activate the non-canonical NFkappaB pathways in trophoblast via RANKL were not successful suggesting that RANKL likely does not play a major role in trophoblast gene regulation. In this study, we show novel regulation of PlGF expression in trophoblast by NFkappaB, which is in part due to modulation of GCM-1 transcriptional activity. We also show that the canonical NFkappaB pathway can be activated in trophoblast by hypoxia. By studying regulation of PlGF in trophoblast by NFkappaB, we may achieve a better understanding of the molecular mechanisms surrounding onset of preeclampsia which may help lead to development of treatment for the disease.