T-cell-derived Interleukin-17 Regulates the Level and Stability of Cyclooxygenase-2 (COX-2) mRNA through Restricted Activation of the p38 Mitogen-activated Protein Kinase Cascade

Abstract

Although interleukin-17 (IL-17) is the pre-eminent Tcell-derived pro-inflammatory cytokine, its cellular mechanism of action remains poorly understood. We explored novel signaling pathways mediating IL-17 induction of the cyclooxygenase-2 (COX-2) gene in human chondrocytes, synovial fibroblasts, and macrophages. In preliminary work, recombinant human (rh) IL-17 stimulated a rapid (5-15 min), substantial (>8-fold), and sustained (>24 h) increase in COX-2 mRNA, protein, and prostaglandin E 2 release. Screening experiments with cell-permeable kinase inhibitors (e.g. SB202190 and p38 inhibitor), Western analysis using specific anti-phospho-antibodies to a variety of mitogen-activated protein kinase cascade intermediates, co-transfection studies using chimeric cytomegalovirus-driven constructs of GAL4 DNA-binding domains fused to the transactivation domains of transcription factors together with Gal-4 binding element-luciferase reporters, ectopic overexpression of activated protein kinase expression plasmids (e.g. MKK3/6), or transfection experiments with wild-type and mutant COX-2 promoter constructs revealed that rhIL-17 induction of the COX-2 gene was mediated exclusively by the stress-activated protein kinase 2/p38 cascade. A rhIL-17-dependent transcriptional pulse (1.76 ؎ 0.11-fold induction) was initiated by ATF-2/CREB-1 transactivation through the ATF/CRE enhancer site in the proximal promoter. However, steadystate levels of rhIL-17-induced COX-2 mRNA declined rapidly (<2 h) to control levels under wash-out conditions. Adding rhIL-17 to transcriptionally arrested cells stabilized COX-2 mRNA for up to 6 h, a process compromised by SB202190. Deletion analysis using transfected chimeric luciferase-COX-2 mRNA 3 -untranslated region reporter constructs revealed that rhIL-17 increased reporter gene mRNA stability and protein synthesis via distal regions (؊545 to ؊1414 bases) of the 3 -untranslated region. This response was mediated entirely by the stress-activated protein kinase 2/p38 cascade. As such, IL-17 can exert direct transcriptional and post-transcriptional control over target proinflammatory cytokines and oncogenes.

Distinct functional motifs within the IL-17 receptor regulate signal transduction and target gene expression

“…IL-17 has been reported to activate various MAPK pathways, and AP1 sites are statistically overrepresented in IL-17 target promoters (35,40,(48)(49)(50). Neither p38 nor JNK were activated by IL-17 in this cell background (data not shown).…”

Identification of Common Transcriptional Regulatory Elements in Interleukin-17 Target Genes

“…IL-17-mediated induction of several genes is due to activation of the proximal promoter as well as enhanced mRNA stability (37)(38)(39)(40). To determine whether IL-17 contributes to 24p3 message levels by enhancing transcript stability, ST2 cells were stimulated with IL-17 for 4 h to induce 24p3 mRNA and then treated with actinomycin D for various time points up to 8 h in the presence or absence of IL-17.…”

Structure–function relationships in the IL-17 receptor: Implications for signal transduction and therapy

“…For example, IL-17 together with TNFα stabilizes IL-6 mRNA [71,[76][77][78]. Similarly, IL-17 serves to enhance mRNA stability of transcripts encoding G-CSF [79], GM-CSF [80], CXCL1/KC/Groα [81], CXCL5/LIX [82,83], IL-8 [77], IκBζ [84] and COX-2 [85,86]. Considerable data implicate MAPK pathways in mRNA stabilization effects.…”

“…Considerable data implicate MAPK pathways in mRNA stabilization effects. In particular, extracellular signal-related kinase p42/44 (ERK) and p38 MAPK are inducibly phosphorylated following IL-17 stimulation, and specific inhibitors to ERK and p38 MAPK reverse enhancement of mRNA stability [76,85,87]. However, other mechanisms are also likely to be involved.…”