Epidermal Growth Factor and Interleukin-1(beta) Utilize Divergent Signaling Pathways to Synergistically Upregulate Cyclooxygenase-2 Gene Expression in Human Amnion-Derived WISH Cells

In human parturition, uterotonic prostaglandins (PGs) arise predominantly via increased expression of cyclooxygenase-2 (COX-2 [also known as prostaglandin synthase 2]) within intrauterine tissues. Interleukin-1 (IL-1) and epidermal growth factor (EGF), both inducers of COX-2 transcription, are among numerous factors that accumulate within amniotic fluid with advancing gestation. It was previously demonstrated that EGF could potentiate IL-1(beta)-driven PGE2 production in amnion and amnion-derived (WISH) cells. To define the mechanism for this observation, we hypothesized that EGF and IL-1(beta) might exhibit synergism in regulating COX-2 gene expression. In WISH cells, combined treatment with EGF and IL-1(beta) resulted in a greater-than-additive increase in COX-2 mRNA relative to challenge with either agent independently. Augmentation of IL-1(beta)-induced transactivation by EGF was not observed in cells harboring reporter plasmids bearing nuclear factor-kappa B (NF(kappa)B) regulatory elements alone, but was evident when a fragment (–891/ +9) of the COX2 gene 5ʹ-promoter was present. Both agents transiently activated intermediates of multiple signaling pathways potentially involved in the regulation of COX-2 gene expression. The 26 S proteasome inhibitor, MG-132, selectively abrogated IL1(beta)-driven NF(kappa)B activation and COX-2 mRNA expression. Only pharmacologic blockade of the p38 mitogenactivated protein kinase eliminated COX-2 expression following EGF stimulation. We conclude that EGF and IL-1(beta) appear to signal through different signaling cascades leading to COX-2 gene expression. IL-1(beta) employs the NF(kappa) B pathway predominantly, while the spectrum of EGF signaling is broader and includes p38 kinase. The synergism observed between IL-1(beta) and EGF does not rely on augmented NF(kappa)B function, but rather, occurs through differential use of independent response elements within the COX-2 promoter.