Nuclear factor kappa B activation and regulation of cyclooxygenase type-2 expression in human amnion mesenchymal cells by interleukin-1β

Abstract

Interleukin-1β (IL-1β) has been shown in numerous studies to increase prostaglandin (PG) output by up-regulating the expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in PG synthesis. In this study, we investigated the possible role of the nuclear factor kappa B (NFκB) in IL-1β signaling, leading to the expression of COX-2 in human amnion cell culture. Fetal amnion was obtained following vaginal delivery and digested with collagenase, and the subepithelial (mesenchymal) cells were isolated. Cultures were characterized with antisera to keratin (epithelial cells) and vimentin (mesenchymal cells). Confluent cells were stimulated with human recombinant IL-1β, and activation of NFκB was assessed by measuring changes in the inhibitory protein IκB (total IκB and phosphorylated IκB) using Western blot analysis as well as by nuclear binding of NFκB using an electrophoretic mobility shift assay. COX-2 protein levels were determined by Western blot analysis. After 5 min of stimulation with IL-1β, phosphorylated IκB began to appear, 90% of which was degraded within 15 min. This was temporally associated with decreased total IκB and increased nuclear NFκB DNA-binding activity. In the IL-1β-treated group, COX-2 protein began to increase after 6 h; this response was time-dependent, with a significant increase until 24 h after IL-1β stimulation. When NFκB translocation was blocked by using SN50 (a cell-permeable inhibitory peptide of NFκB translocation), the synthesis of COX-2 protein was inhibited. These results suggest that NFκB is involved in the IL-1β-induced COX-2 expression in the mesenchymal cells of human amnion.