| 研究概要 |
FY2013 To clarify the TLR4-mediated signaling pathway involved in regulating Arid5a Expression
Post-transcriptional regulation of IL-6 has been largely uncharacterized, with the exception of the RNase Regnase-l, which prevents autoimmunity by destabilizing IL-6 mRNA. Recently, we identified a novel RNA binding protein, AT-rich interactive domain 5a (Arid5a), which stabilizes IL-6 but not TNF-a mRNA through binding to the 3' un-translated region (UTR) of IL-6 mRNA. Signaling through P38-a MAP kinase is involved in stabilization of IL-6 mRNA in mouse embryonic fibroblasts. P38 signaling is critically required in dendritic cells (not macrophage or T cells) for the induction of Th17 cells in experimental autoimmune encephalitis (EAE), a murine model of multiple sclerosis. However, the key molecules through P38 MAP kinase signaling remain uncharacterized. The identification of such molecules is of importance since they may represent potential therapeutic targets for IL-6 blockade. Interestingly, we recently found that chlorpromazine (an anti-histaminic/anti-psychotic drug), which inhibits P38-a MAP kinase activity, inhibited LPS-induced Arid5A expression and selectively inhibited LPS-induced IL-6 production in cultured macrophages. In the current study (FY2013) we have investigated if P38 MAP kinase signaling is linked to the induction of IL-6 mRNA stabilization and induction of Th17 cells through induction of Arid5A.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
Confirmation that P38 MAP kinase signaling pathway regulates stabilization of Arid5a mRNA.
In the current study (FY2013), we observed that through TLR4 signaling, Arid5a mRNA is induced rapidly and degrades quickly in macrophages, B cells and dendritic cells. Interestingly, we found that through TLR4 signaling, chemical inhibitors of P38 MAP kinase, significantly enhanced the degradation of Arid5a mRNA levels in macrophages, B cells and dendritic cells. Thus our data indicates that P38 MAP kinase signaling enhances the mRNA stability of Arid5a, which in-turn leads to IL-6 mRNA stabilization. Importantly therefore we have been able to confirm our preliminary hypothesis that Arid5a represents a key molecule by which P38 MAP kinase signaling functions to stabilize IL-6 mRNA.
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| 今後の研究の推進方策 |
Our current research data indicates an important role for p38 MAP kinase signaling pathway in the post-transcriptional regulation of Arid5a expression. My continuing studies will therefore focus on the mechanisms by which P38 MAP kinase pathway enhances stabilization of Arid5a mRNA transcripts, and on the role of Arid5a in mediating the known roles of P38 MAP kinase in stabilization of IL-6 mRNA and induction of Th17 cells in autoimmunity.
i) Molecular mechanisms of p38 MAP kinase stabilization of Arid5a mRNA.
First we will extend our previous analysis (using chemical inhibitors) of P38 MAP kinase pathway involvement in Arid5a mRNA stabilization using immune cells (macrophages, dendritic, B cells) obtained from P38 knockout mice. Then, having clearly established the role of P38 MAP kinase signaling on Arid5a mRNA stabilization, we will investigate the molecular mechanisms. Our preliminary hypothesis is that this pathway may induce the binding of specific proteins to Arid5a mRNA, which in
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-turn confers stabilization of the RNA molecule. For this procedure we will perform immune-affinity purification and mass-spectrometric analysis of Arid5a mRNA-binding proteins from cytoplasmic extracts of LPS-stimulated macrophages from WT mice and P38 KO mice.
ii) Role of Arid5a in P38 MAP kinase pathway regulation of IL-6 mRNA stability and Th17 cell induction.
Although P38 MAP kinase signaling is known to regulate IL-6 mRNA stability and Th17 cell induction, the key molecule (s) which mediate this effect have not been characterized. Arid5a may represent this key molecule, since our new data shows that P38 MAP kinase signaling regulates Arid5a expression, while our recent published study demonstrated a role for Arid5a in IL-6 mRNA stabilization and Th17 induction.
(a) We will utilize WT, P38 knockout mice, Arid5a transgenic mice and P38 (-/-)/Arid5aTg mice. Using macrophages, B cells and dendritic cells obtained from these mice, we will conduct in vitro analyses to determine if Arid5a overexpression rescues impairment of IL-6 stabilization in P38 KO cells following LPS stimulation.
b) Experimental autoimmune encephalitis (EAE) is a murine model of multiple sclerosis. EAE is an IL-6-dependent disease, in which IL-6 production through P38 MAP kinase signaling in dendritic cells is required for Th17 induction and disease. Thus, following induction of EAE in WT, P38 knockout mice, Arid5a transgenic mice and P38 (-/-)/Arid5aTg mice, we will determine if overexpression of Arid5a rescues impairment of IL-6 production in dendritic cells and impairment of Th17 cell development. 隠す
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