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-kappa B) 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-alpha and LPS activate the NF-kappa B pathway; and (ii) to use short interfering RNA (siRNA) against inhibitor kappa B kinase (IKK)-beta to confirm the role of the NF-kappa B 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 mu g/ml LPS or 20 ng/ml TNF-alpha, or (ii) transfected with 100 nM IKK-beta siRNA. Incubation of JEG-3 cells with LPS and TNF-alpha increased the expression of cytoplasmic IKK-beta and phosphorylated I kappa B-alpha, and nuclear NF-kappa B proteins p50 and p65. This was associated with a concurrent increase in COX-2 protein, and IL-6 and PGF(2 alpha) release from JEG-3 cells. Treatment of cells with BAY 11-7082 at 50 mu M significantly inhibited basal, LPS- and TNF-alpha-induced NF-kappa B and COX-2 expression, and IL-6 and PGF(2 alpha) release. Transfection of JEG-3 cells with IKK-beta siRNA significantly decreased IL-6 and PGF(2 alpha) release. The data presented in this study demonstrate that pro-inflammatory mediators regulate the NF-kappa B transcription pathway in human JEG-3 cells, and the IKK-beta/NF-kappa B pathway is a regulator of inflammatory mediators in placental JEG-3 cells.