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. 2018 Feb 6;115(6):E1214-E1220.
doi: 10.1073/pnas.1719921115. Epub 2018 Jan 22.

Regulation of inflammatory responses by dynamic subcellular localization of RNA-binding protein Arid5a

Mitsuru Higa  1   2 , Masahiro Oka  2 , Yoshitaka Fujihara  3 , Kazuya Masuda  1 , Yoshihiro Yoneda  4 , Tadamitsu Kishimoto  5
Affiliations

Regulation of inflammatory responses by dynamic subcellular localization of RNA-binding protein Arid5a

Mitsuru Higa et al. Proc Natl Acad Sci U S A. .

Abstract

Adenine-thymine (AT)-rich interactive domain 5a (Arid5a) is an RNA-binding protein found in the cytoplasm and nucleus of normally growing cells. Although Arid5a is known to play an important role in immune regulation, whether and how Arid5a subcellular localization impacts immune regulation has remained unclear. In this study, we generated Arid5a transgenic (TG) mice to address this question. While ectopic Arid5a overexpression did not affect expression of inflammatory cytokines under unstimulated conditions, significantly higher levels of inflammatory cytokines, such as IL-6, were produced in response to lipopolysaccharide (LPS) stimulation. Consistent with this, TG mice were more sensitive to LPS treatment than wild-type mice. We also found that Arid5a is imported into the nucleus via a classical importin-α/β1-mediated pathway. On stimulation, nuclear Arid5a levels were decreased, while there was a concomitant increase in cytoplasmic Arid5a. Arid5a is associated with up-frameshift protein 1, and its nuclear export is regulated by a nuclear export receptor, chromosomal region maintenance 1. Taken together, these data indicate that Arid5a is a dynamic protein that translocates to the cytoplasm from the nucleus so as to properly exert its dual function in mRNA stabilization and transcriptional regulation during inflammatory conditions.

Keywords: Arid5a; IL-6; LPS; cytoplasm; inflammation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Arid5a did not affect inflammatory cytokine production under unstimulated conditions. (A–C) qRT-PCR analysis of Arid5a (A), Il6 (B), and Tnf (C) mRNAs in peritoneal macrophages, normalized to the expression of GAPDH. (D and E) Serum levels of IL-6 (D) and TNF (E) in WT and Arid5a TG mice were measured by ELISA. Data show the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences between WT and TG values. **P < 0.01, Student’s t test. N.D., not detected.
Fig. 2.
Fig. 2.
Arid5a TG mice were more sensitive to endotoxic shock than WT mice. (A) The 6- to 8-wk-old WT (n = 10) and Arid5a TG mice (n = 10) were injected with E. coli LPS (12.5 mg/kg), and survival was monitored for 96 h. (B and C) WT and Arid5a TG mice (6–8 wk old) were injected with LPS (5 mg/kg). Serum levels of IL-6 (B) or TNF (C) measured by ELISA at the indicated time points after LPS challenge. Data are the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences between WT and TG values (*P < 0.05, Student’s t test).
Fig. 3.
Fig. 3.
Arid5a was actively imported into the nucleus. (A) Immunofluorescence staining of RAW264.7 cells was performed. The cells were incubated with indicated antibodies, and nuclei were visualized with DAPI. (Scale bar: 10 µm.) (B) HEK293T cells were transfected with Flag-tagged Arid5A or empty vector. Whole-cell lysates were prepared. Immunoprecipitation was performed with anti-Flag beads, followed by Western blotting with anti-Flag and anti-KPNA2 antibodies. (C) RAW264.7 cells were transfected with an empty or bimax vector. After 24 h, the cells were fractionated into cytoplasmic and nuclear extracts and analyzed by Western blot analysis using the indicated antibodies. (D) RAW264.7 cells were transfected with empty or bimax vector. After 24 h, cells were subjected to immunofluorescence staining. The cells were incubated with anti-Arid5a antibodies. The nuclei were visualized with DAPI. (Scale bar: 10 µm.)
Fig. 4.
Fig. 4.
Nucleocytoplasmic ratio of Arid5a protein changes after LPS stimulation (A) RAW264.7 cells were stimulated with or without LPS (500 ng/mL) for 4 h and then analyzed by immunofluorescence staining. The cells were incubated with anti-Arid5a antibodies, and nuclei were visualized with DAPI. (Scale bar: 10 µm.) (B) Mean nuclear/cytoplasm ratios (n = 50; **P < 0.01; Student’s t test). (C and D) Cells were stimulated with LPS (RAW264.7 cells: 500 ng/mL, peritoneal macrophages: 100 ng/mL) at the indicated times and were then fractionated into cytoplasmic and nuclear extracts and analyzed by Western blot analysis using the indicated antibodies. (E) RAW264.7 cells were stimulated with LPS (100 ng/mL) at the indicated times. Cytoplasmic and nuclear extracts were isolated, and Il6 mRNA was evaluated by qRT-PCR, with normalization to GAPDH expression. The data show the mean ± SD of three independent experiments. Asterisks indicate statistically significant differences between time point values (*P < 0.05; **P < 0.01, Student’s t test). (F) After LPS stimulation for 4 h, total RNA isolated from Arid5a immunoprecipitation of each compartment was subjected to qRT-PCR analysis. RNA levels in the Arid5a-bound fraction of each compartment were normalized to input levels and then compared with those in cells without stimulation. Data show the mean ± SD of three independent experiments. Asterisks indicate statistically significant differences between unstimulated and stimulated Arid5a-Il6 mRNA values (**P < 0.01, Student’s t test).
Fig. 5.
Fig. 5.
Arid5a was associated with UPF1. (A) RAW264.7 cells were lysed with NETN buffer, and whole-cell lysates were then prepared. Immunoprecipitation was performed with anti-Arid5a antibodies and protein G Sepharose, followed by Western blot analysis with the indicated antibodies. (B) Schematic representation of truncated Arid5A. The Arid domain is shown in red. The amino acids present in each mutant are indicated. (C) Flag-tagged full-length (1–589) or truncated (1–293, 1–450, or 150–589) Arid5a was expressed with Myc-tagged UPF1 in HEK293T cells, and cell lysates were prepared. Immunoprecipitation was performed with anti-Flag beads, followed by Western blot analysis with anti-Flag and anti-Myc antibodies. (D) RAW264.7 cells were treated with or without LMB (10 nM) for 2 h, LPS (500 ng/mL) for 4 h, or LPS for 2 h + LMB for 2 h, and then analyzed by immunofluorescence staining. The nuclei were visualized with DAPI. (Scale bar: 10 µm.) (E) Fluorescence intensity of nuclear Arid5a. Asterisks indicate statistically significant differences between samples (n = 50; **P < 0.01; Student’s t test). (F) Levels of IL-6 in the medium from cultures of RAW264.7 cells were measured by ELISA. Data show the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences between LPS and LPS → LMB values (**P < 0.01, Student’s t test).
Fig. 6.
Fig. 6.
Model of inflammatory response by Arid5a/Regnase-1/UPF1. Regnase-1 localizes to the cytoplasm. Arid5a is imported via an importin-α/β1 pathway. UPF1 shuttles between the nucleus and the cytoplasm. In response to inflammation, the Toll-like receptor (TLR) is activated by LPS, and Il6 mRNA is induced by NF-κB. After that, Arid5a interacts with Il6 mRNA. Arid5a is exported to the cytoplasm via CRM1 pathway with UPF1.
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