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We investigated the regulation of PG production in human endometrial stromal cells (ESC) by IL-1beta. We found that cyclooxygenase-2 (COX-2) mRNA and protein levels and PGE(2) production in ESC were significantly increased by IL-1beta. COX-2 mRNA, protein, and PGE(2) levels in IL-1beta-treated ESC were decreased by a PKA inhibitor, a nuclear factor (NF-kappaB) inhibitor, and an ERK1/2 inhibitor, but not by a p38 MAPK inhibitor or a PKC inhibitor, suggesting the possible involvement of PKA, NF-kappaB, and/or the ERK1/2 signaling pathway(s) in IL-1beta-mediated COX-2 gene induction in ESC. We then transiently transfected deletion mutants of the COX-2 promoter fused to the luciferase reporter gene and variants of -360/+56 bp promoter construct carrying different site-directed mutations of selected cis-acting elements. We determined that a NF-kappaB site (-222/-213 bp), a nuclear factor for IL-6 expression site (NF-IL6, -132/-124 bp), and a cAMP response element (-59/-52 bp) were essential for the baseline COX-2 gene promoter regulation. The addition of IL-1beta, however, did not affect the activity of these COX-2 promoter constructs. To investigate the potential effects of IL-1beta on COX-2 mRNA stability, ESC were treated with actinomycin D, a general transcription inhibitor, in the absence or presence of IL-1beta. We found that 1) IL-1beta significantly increased COX-2 mRNA stability; 2) continuous transcription was not required to sustain the IL-1beta-induced COX-2 mRNA levels; and 3) COX-2 mRNA was highly unstable in the absence of IL-1beta. Additionally, we found that the ERK1/2 signaling pathway was essential for stabilizing COX-2 mRNA. We conclude that levels of COX-2 mRNA, protein, and enzyme activity in ESC are controlled by various signaling pathways, including PKA, ERK1/2, and NF-kappaB. Moreover, posttranscriptional mRNA stability is an important mechanism for IL-1beta-induced elevation of COX-2 expression in ESC.

the journal of clinical endocrinology and metabolism/journal of clinical endocrinology and metabolism

Interleukin-1β Elevates Cyclooxygenase-2 Protein Level and Enzyme Activity via Increasing Its mRNA Stability in Human Endometrial Stromal Cells: An Effect Mediated by Extracellularly Regulated Kinases 1 and 2