Lipopolysaccharide and TNF-α Activate the Nuclear Factor Kappa B Pathway in the Human Placental JEG-3 Cells

Up-regulation of pro-inflammatory cytokines, cyclooxygenase (COX-2) and prostaglandins is a critical factor driving human term labour and inflammation-associated preterm labour. Nuclear factor kappa B (NF-κB) is activated in response to a number of inflammatory mediators, including cytokines and lipopolysaccharide (LPS). The aim of this study was (i) to investigate if TNF-α and LPS activate the NF-κB pathway; and (ii) to use short interfering RNA (siRNA) against inhibitor κB kinase (IKK)-β to confirm the role of the NF-κB pathway in the regulation of pro-inflammatory mediators in human placental JEG-3 cells. JEG-3 cells (3 independent experiments) were (i) incubated in the presence or absence of 10 μg/ml LPS or 20 ng/ml TNF-α, or (ii) transfected with 100 nM IKK-β siRNA. Incubation of JEG-3 cells with LPS and TNF-α increased the expression of cytoplasmic IKK-β and phosphorylated IκB-α, and nuclear NF-κB proteins p50 and p65. This was associated with a concurrent increase in COX-2 protein, and IL-6 and PGF 2α release from JEG-3 cells. Treatment of cells with BAY 11-7082 at 50 μM significantly inhibited basal, LPS- and TNF-α-induced NF-κB and COX-2 expression, and IL-6 and PGF 2α release. Transfection of JEG-3 cells with IKK-β siRNA significantly decreased IL-6 and PGF 2α release. The data presented in this study demonstrate that pro-inflammatory mediators regulate the NF-κB transcription pathway in human JEG-3 cells, and the IKK-β/NF-κB pathway is a regulator of inflammatory mediators in placental JEG-3 cells.