Leukotriene B4 BLT Receptor Signaling Regulates the Level and Stability of Cyclooxygenase-2 (COX-2) mRNA through Restricted Activation of Ras/Raf/ERK/p42 AUF1 Pathway

摘要

Recent studies suggest that active resolution of the inflammatory response in animal models of arthritis may involve leukotriene B4 (LTB4)-dependent stimulation of “intermediate” prostaglandin production, which in turn favors the synthesis of “downstream” anti-inflammatory and pro-resolving lipoxins, resolvins, and protectins. We explored a putative mechanism involving LTB4-dependent control of cyclooxygenase-2 (COX-2) expression, the rate-limiting step in inflammatory prostaglandin biosynthesis. Indeed, LTB4 potently up-regulated/stabilized interleukin-1β-induced COX-2 mRNA and protein expression under conditions of COX-2 inhibitor-dependent blockade of PGE2 release in human synovial fibroblasts (EC50 = 16.5 ± 1.7 nm for mRNA; 19 ± 2.4 nm for protein, n = 4). The latter response was pertussis toxin-sensitive, and semi-quantitative reverse transcription-PCR confirmed the quantitative predominance of the BLT2 receptor. Transfection experiments, using human COX-2 promoter plasmids and chimeric luciferase-COX-2 mRNA 3′-untranslated region (3′-UTR) reporter constructs, revealed that LTB4 exerted its stabilizing effect at the post-transcriptional level through a 116-bp adenylate/uridylate-rich sequence in the proximal region of the COX-2 3′-UTR. Using luciferase-COX-2 mRNA 3′-UTR reporter constructs and Ras/c-Raf expression and mutant constructs, we showed that the Ras/c-Raf/MEK1/2/ERK1/2 signaling pathway mediated LTB4-dependent COX-2 mRNA stabilization. Knockdown experiments with specific short hairpin RNAs confirmed that LTB4 stabilization of COX-2 mRNA was apparently mediated through the RNA-binding protein, p42 AUF1. The nuclear export of p42 AUF1 was driven by c-Raf/MEK1/2/ERK1/2 signaling and sensitive to leptomycin B treatment, suggesting a CRM1-dependent mechanism. We conclude that LTB4 may support the resolution phase of the inflammatory response by stabilizing COX-2, ensuring a reservoir of ambient pro-resolution lipid mediators.