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Regular Article |
a Division of Maternal-Fetal Medicine and Reproductive Biology Unit, Department of Obstetrics and Gynecology and Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa Health Research Institute, Ottawa, Ontario, Canada K1H 8L6
Interleukin-1ß (IL-1ß) 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 (NF
B) in IL-1ß 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-1ß, and activation of NFB was assessed by measuring changes in the inhibitory protein IB (total IB and phosphorylated IB) using Western blot analysis as well as by nuclear binding of NFB using an electrophoretic mobility shift assay. COX-2 protein levels were determined by Western blot analysis. After 5 min of stimulation with IL-1ß, phosphorylated IB began to appear, 90% of which was degraded within 15 min. This was temporally associated with decreased total IB and increased nuclear NFB DNA-binding activity. In the IL-1ß-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-1ß stimulation. When NFB translocation was blocked by using SN50 (a cell-permeable inhibitory peptide of NFB translocation), the synthesis of COX-2 protein was inhibited. These results suggest that NFB is involved in the IL-1ß-induced COX-2 expression in the mesenchymal cells of human amnion.
1 Supported by CIHR (grant MOP-37943 to W.G.) and the Chinese Education Commission (scholarship to X.Y.).
2 Correspondence: William Gibb, Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Ottawa Hospital-General Campus, 501 Smyth Rd., Ottawa, ON, Canada K1H 8L6. FAX: 613 737 8070/8470; wgibb{at}ottawahospital.on.ca or wgibb{at}attcanada.ca
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