Expression of COX-2 in platelet-monocyte interactions occurs via combinatorial regulation involving adhesion and cytokine signaling
Dan A. Dixon, … , Stephen M. Prescott, Guy A. Zimmerman
Dan A. Dixon, … , Stephen M. Prescott, Guy A. Zimmerman
Published October 2, 2006
Citation Information: J Clin Invest. 2006;116(10):2727-2738. https://doi.org/10.1172/JCI27209.
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Research Article Inflammation

Expression of COX-2 in platelet-monocyte interactions occurs via combinatorial regulation involving adhesion and cytokine signaling

Abstract

Tight regulation of COX-2 expression is a key feature controlling eicosanoid production in atherosclerosis and other inflammatory syndromes. Adhesive interactions between platelets and monocytes occur in these conditions and deliver specific signals that trigger inflammatory gene expression. Using a cellular model of monocyte signaling induced by activated human platelets, we identified the central posttranscriptional mechanisms that regulate timing and magnitude of COX-2 expression. Tethering of monocytes to platelets and to purified P-selectin, a key adhesion molecule displayed by activated platelets, induces NF-κB activation and COX-2 promoter activity. Nevertheless, COX-2 mRNA is rapidly degraded, leading to aborted protein synthesis. Time-dependent signaling of monocytes induces a second phase of transcript accumulation accompanied by COX-2 enzyme synthesis and eicosanoid production. Here, generation of IL-1β, a proinflammatory cytokine, promoted stabilization of COX-2 mRNA by silencing of the AU-rich mRNA decay element (ARE) in the 3′-untranslated region (3'UTR) of the mRNA. Consistent with observed mRNA stabilization, activated platelets or IL-1β treatment induced cytoplasmic accumulation and enhanced ARE binding of the mRNA stability factor HuR in monocytes. These findings demonstrate that activated platelets induce COX-2 synthesis in monocytes by combinatorial signaling to transcriptional and posttranscriptional checkpoints. These checkpoints may be altered in disease and therefore useful as targets for antiinflammatory intervention.

Authors

Dan A. Dixon, Neal D. Tolley, Kristi Bemis-Standoli, Mark L. Martinez, Andrew S. Weyrich, Jason D. Morrow, Stephen M. Prescott, Guy A. Zimmerman

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Figure 3

Adhesion of monocytes to P-selectin induces rapid COX-2 transcription.

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Adhesion of monocytes to P-selectin induces rapid COX-2 transcription. (...
(A) Monocytes (106 cells) were incubated on plates coated with immobilized P-selectin, and total RNA was prepared at the indicated times (h). COX-2 mRNA was detected by RNase protection assay using 28S RNA as a control for RNA loading. (B) Adhesion to P-selectin induces COX-2 promoter activity in U937 cells. A LUC reporter construct containing the 1.8-kb COX-2 promoter was transfected into U937 cells, which were subsequently kept in suspension (white bars) or allowed to adhere to purified, immobilized P-selectin (black bars) for 48 hours. In parallel, NF-κB promoter activity was determined using a LUC reporter construct containing 5 consensus NF-κB–binding elements. The pGL3 control vector containing the SV40 promoter and enhancer elements was used to evaluate specificity of P-selectin–mediated transcriptional induction. Relative activity was assessed as LUC activity normalized to total protein. All values shown are normalized to expression of LUC by transfected cells in suspension and indicate the averages of 3 experiments. (C) COX-2 mRNA t1/2 in monocytes adherent to immobilized P-selectin. ActD (5 μg/ml) was added after monocytes had been incubated in P-selectin–coated plates for 1 hour, and total RNA was prepared at the indicated time points. COX-2 mRNA decay was analyzed by Northern blot probing for COX-2. 18S RNA is shown as a control for RNA loading. The blot shown is from 1 of 2 experiments with similar results.