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

Higa, Mitsuru, Oka, Masahiro, Fujihara, Yoshitaka, Masuda, Kazuya, Yoneda, Yoshihiro, Kishimoto, Tadamitsu·2018·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.1719921115PMC PMC5819453
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Abstract

Significance: Immune cell activation is accompanied by dynamic changes in expression of genes related to inflammation. Concomitantly, immune reactions are tightly controlled to prevent harmful pathologies due to sustained inflammation. Gene expression is controlled at multiple checkpoints. Among these, the post-transcriptional regulation of the balance between Arid5a and Regnase-1 is important for the induction of inflammation. Our findings provide important insight into the role of the regulation of nucleocytoplasmic localization of Arid5a in the development of inflammation through the induction of a change in the ratio of Arid5a to Regnase-1.

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Figures

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. 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.CC BY-NC-ND · PubMed Central
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. 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).CC BY-NC-ND · PubMed Central
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. 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.)CC BY-NC-ND · PubMed Central
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. 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).CC BY-NC-ND · PubMed Central
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. 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).CC BY-NC-ND · PubMed Central
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.
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.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Higa, M., Oka, M., Fujihara, Y., Masuda, K., Yoneda, Y., & Kishimoto, T.
Affiliations
  • Laboratory of Immune Regulation, Immunology Frontier Research Center, Osaka University, Suita, 565-0871 Osaka, Japan;
  • Laboratory of Nuclear Transport Dynamics, National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki, 567-0085 Osaka, Japan;
  • Research Institute for Microbial Diseases, Osaka University, Suita, 565-0871 Osaka, Japan;
  • National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki, 567-0085 Osaka, Japan
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
2018-01-22
Volume
115
Issue
6
ISSN
  • 0027-8424
  • 1091-6490
DOI
10.1073/pnas.1719921115
PMC
PMC5819453
Cited by
61
References
34
Language
en
Licence
creativecommons.org/licenses/by-nc-nd/4.0/ (vor)

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