P079 ARID5a is an IL-6 mRNA stability protein. Chlorpromazine mediates its inhibitory effect on IL-6 production in macrophages through inhibition of ARID5A expression

Abstract

Introduction Our previous study showed that chlorpromazine (CPZ), an anti-histamine therapeutic, inhibits LPS-induced IL-6 but not TNF-α production in macrophages [1] . In the current study, we show that CPZ is an inhibitor of LPS-induced IL-6 but not TNF-α mRNA stability. Since the 3′-untranslated region (3′-UTR) is important for the regulation of IL-6 mRNA stability, we examined whether CPZ affects the activity of RNA stabilizing proteins on AU-rich IL-6 3′-UTR mRNA. Here we identified a novel IL-6 mRNA stability protein, Arid5a. The Arid (AT-rich interactive domain) family of proteins have been previously demonstrated to be involved in the control of differentiation, tissue-specific gene expression and proliferation control [2] . In particular Arid5a was shown to be involved in chondrocyte-specific gene transcription [3] . In this study, we report a novel function for Arid5a on the stabilization of IL-6 mRNA. Methods For knockdown of Arid5a gene expression we employed siRNA technology. Levels of IL-6 and TNF-alpha protein in RAW 264 macrophage cell supernatants following transfection of siRNA control or ARID5a, were determined by ELISA. IL-6 and TNF-alpha mRNA stability was determined by real-time PCR. Interaction of ARID5a with the IL-6 3′-UTR was examined by mass spectrometry (LC-MS/MS). Results We found that Arid5a is induced by LPS stimulation in macrophages and binds to the IL-6 3′-UTR in macrophages. In RAW 264 cells, knockdown of Arid5a by siRNA led to the decrease of LPS-induced IL-6 but not TNF-α production. We showed that overexpression of Arid5a critically stabilizes IL-6 mRNA through AU-rich elements of the 3′-UTR region. Conclusion Thus, Arid5a is an IL-6 mRNA stability protein which mediates the inhibitory effects of CPZ on LPS-mediated IL-6 production. CPZ, or novel derivatives of this molecule, may represent a new therapy for IL6-dependent diseases (such as arthritis), through its inhibitory function on IL-6 mRNA stability.