Background/Purpose: Changes in gene expression are closely related to inflammation and joint destruction in RA. Recently, it has been recognized that post-transcriptional regulation of the gene expression of various cytokines is important to the immunological process. In this study, we attempted to identify cytokines that regulate the metabolism of RA-related mRNA through unique AU rich elements (ARE), which are characterized by AUUUA repeats in their 3′ untranslated regions (3′UTR).

Methods: We generated dual luciferase reporter plasmids to study the influence of cytokines on the stability of transcripts associated with RA. The 3′-UTR sequences of RA-related genes (CXCL2, PIM1, CSF2, IL12B, IL1B, TNF, TNFRSF10B, HIF1A, ARHGEF2) with ARE were inserted into the 3′ end of the firefly luciferase gene in the pmirGLO Dual-Luciferase miRNA Target Expression Vector. Renilla luciferase in the plasmid was used as a control reporter for normalization. U2OS cells were transfected with these plasmids 2 hours before cytokine treatment. The transfected cells were stimulated with cytokines for 12 hours and were lysed using a passive lysis buffer for luciferase activity analyses with the Dual-Glo Luciferase Assay System and Infinite 200 PRO. TIA1, TIAR, TTP, or HuR were co-transfected with the reporter. The expression of CXCL2 mRNA in fibroblast-like synoviocytes (FLS) was examined by RT-PCR. In mRNA decay experiments, actinomycin D was used to stop RNA polymerase II dependent transcription in FLS.

Results: Various cytokine treatments affected the reporter luciferase activities. In particular, IL-1β and IL-17A increased the luciferase activities of the reporters harboring the 3′-UTR of CXCL2 mRNA in a dose-dependent manner. Addition of IL-1β remarkably upregulated the CXCL2 mRNA expression in FLS. The half-life of CXCL2 mRNA was significantly longer in IL-1β treated FLS (21.7 hours) than that in untreated FLS (3.3 hours). ARE-harboring mRNA is potentially regulated by RNA binding proteins including TIA1, TIAR, TTP, and HuR. Among these, co-transfection of TTP with the reporter plasmid suppressed the luciferase activity with the 3′UTR of CXCL2 mRNA.

Conclusion: Our data suggest that the metabolism of CXCL2 mRNA is stabilized by IL-1β, leading to a remarkable increase in CXCL2 mRNA in FLS. CXCL2 is a chemokine that is secreted from various cells for chemotactic attraction of polymorphonuclear leukocytes and is involved in the pathogenesis of infections and autoimmune diseases. Therefore, it is possible that IL-1β signaling stabilizes CXCL2 mRNA by regulating RNA binding proteins like TTP, for the recruitment of neutrophils at the inflamed joint.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/interleukin-1%ce%b2-stabilize-cxcl2-mrna-and-increase-its-expression/