Expression of COX-2 in platelet-monocyte interactions occurs via combinatorial regulation involving adhesion and cytokine signaling
Dan A. Dixon, Neal D. Tolley, Kristi Bemis-Standoli, Mark L. Martinez, Andrew S. Weyrich, Jason D. Morrow, Stephen M. Prescott, Guy A. Zimmerman
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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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 8

IL-1β regulates COX-2 mRNA decay through the ARE-binding protein HuR.

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IL-1β regulates COX-2 mRNA decay through the ARE-binding protein HuR. (A...
(A) LUC/3'UTR-reporter constructs containing no 3'UTR (LUCΔ3'UTR, white bars) or COX-2 3'UTR (LUC+3'UTR, black bars) were transfected into U937 cells. LUC activity (RLU) was normalized to total protein and is the average of 3 experiments. (B) U937 cells were transfected with LUC/3'UTR reporter constructs and kept in suspension medium (co), incubated on immobilized P-selectin, kept in suspension in medium containing 10 ng/ml IL-1β, or incubated on immobilized P-selectin in medium containing IL-1β. Relative LUC activity was normalized to total protein, and values shown are based on LUC expression from control transfections. (C) U937 cells (106 cells) were left untreated (co) or incubated for 18 hours with 10 ng/ml IL-1β. ARE-binding proteins HuR and TIA-1 were examined in 15 μg of total cell lysates and 25 μg of cytoplasmic lysates by immunoblot. β-actin was detected as a loading control. (D) Cytoplasmic lysates from untreated (co) or IL-1β–treated U937 cells were incubated with 32P-labeled AREs based on COX-2 or GM-CSF mRNA sequences. Bound proteins were cross-linked to the ARE and immunoprecipitated, using anti-HuR Ab or control IgG. The arrowhead indicates the major immunoprecipitated species. (E) Platelets were incubated with monocytes for 1 hour and 18 hours in the presence of thrombin, then examined for HuR localization by immunocytochemical analysis. Immunofluorescence of HuR in platelet-monocyte aggregates is shown in green; platelet and monocyte cytoskeleton are shown in red. Thick arrows indicate monocyte nuclei; thin arrow indicates monocyte cytoplasm. Scale bar: 5 μm.