Lipopolysaccharide and TNF-alpha 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-kappaB) 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-kappaB pathway; and (ii) to use short interfering RNA (siRNA) against inhibitor kappaB kinase (IKK)-beta to confirm the role of the NF-kappaB 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 microg/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 IkappaB-alpha, and nuclear NF-kappaB proteins p50 and p65. This was associated with a concurrent increase in COX-2 protein, and IL-6 and PGF2alpha release from JEG-3 cells. Treatment of cells with BAY 11-7082 at 50 microM significantly inhibited basal, LPS- and TNF-alpha-induced NF-kappaB and COX-2 expression, and IL-6 and PGF2alpha release. Transfection of JEG-3 cells with IKK-beta siRNA significantly decreased IL-6 and PGF2alpha release. The data presented in this study demonstrate that pro-inflammatory mediators regulate the NF-kappaB transcription pathway in human JEG-3 cells, and the IKK-beta/NF-kappaB pathway is a regulator of inflammatory mediators in placental JEG-3 cells.
Lappas
M
M
Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, 126 Clarendon Street, East Melbourne, Victoria 3002, Australia. mlappas@unimelb.edu.au
Yee
K
K
Permezel
M
M
Rice
G E
GE
D045744:Cell Line, Tumor; D002822:Choriocarcinoma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D051546:Cyclooxygenase 2 / Q000378:metabolism; D015237:Dinoprost / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D006801:Humans; D051550:I-kappa B Kinase / Q000235:genetics / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008565:Membrane Proteins / Q000378:metabolism; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D009570:Nitriles / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D013450:Sulfones / Q000494:pharmacology; D014162:Transfection; D014327:Trophoblasts / Q000187:drug effects / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
C434003:3-(4-methylphenylsulfonyl)-2-propenenitrile; D004338:Drug Combinations; D015850:Interleukin-6; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D016328:NF-kappa B; D009570:Nitriles; D034741:RNA, Small Interfering; D013450:Sulfones; D014409:Tumor Necrosis Factor-alpha; D015237:Dinoprost; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D051550:I-kappa B Kinase