Arid5a controls IL-6 mRNA stability, which contributes to elevation of IL-6 level in vivo

Abstract

Posttranscriptional regulation of IL-6 has been largely uncharacterized, with the exception of the ribonuclease Regnase-1, which prevents autoimmunity by destabilizing IL-6 mRNA. Here, we identified AT-rich interactive domain-containing protein 5A (Arid5a) as a unique RNA binding protein, which stabilizes IL-6 but not TNF-α mRNA through binding to the 3' untranslated region of IL-6 mRNA. Arid5a was enhanced in macrophages in response to LPS, IL-1β, and IL-6. Arid5a deficiency inhibited elevation of IL-6 serum level in LPS-treated mice and suppressed IL-6 levels and the development of T(H)17 cells in experimental autoimmune encephalomyelitis. Importantly, Arid5a inhibited the destabilizing effect of Regnase-1 on IL-6 mRNA. These results indicate that Arid5a plays an important role in promotion of inflammatory processes and autoimmune diseases.

Keywords: RNA-protein complex; immune regulation.

Fig. 1.

Identification of a unique RNA-Bp, Arid5a, on the IL-6 3′ UTR. (A) Peritoneal macrophages were stimulated with LPS (1 μg/mL) for 2 h with or without CPZ (20 μM). Whole-cell lysates were mixed with the IL-6 or TNF-α 3′ UTR (containing BrU) binding protein G beads and then eluted buffer were subjected to SDS/PAGE, respectively. (B) The gel from the complex of the IL-6 3′ UTR and beads (as in A) was stained by CBB after SDS/PAGE and separated three compartments were analyzed by LC-MS/MS. The detected bands were indicated at arrows. (C) The samples from lysates (as in A) were used as input (β-actin detection by immunoblotting). After SDS/PAGE (as in A), endogenous Arid5a was detected with anti-Arid5a antibody by immnoblot analysis. (D) Arid5a recombinant protein or BSA was mixed with the IL-6 3′ UTR (containing BrU) binding protein G beads and then eluted buffer were subjected to SDS/PAGE, respectively. The samples were analyzed by immunoblotting using anti-Arid5a or anti-BSA antibody (Fig. S3). (E) Total RNAs at the indicated times were collected from peritoneal macrophages stimulated by LPS (1 μg/mL), and Arid5a mRNA levels were measured by qPCR. (F) Peritoneal macrophages were treated with LPS (1 μg/mL) at the indicated times. The cells were lysed and analyzed by immunoblotting. Endogenous Arid5a was detected with anti-Arid5a antibody. (G) Arid5a mRNA levels in peritoneal macrophages were compared 2 h after LPS (100 ng/mL) stimulation with or without CPZ (50 μM) pretreatment for 30 min. (H) Peritoneal macrophages were treated 2 h with LPS (1 μg/mL) with or without CPZ (20 μM) pretreatment. The cells were lysed and analyzed by Immunoblotting. (C, D, F, and H) Data are representative of three independent experiments. (E and G) Data show means ± SD of three independent experiments. ***P < 0.001 (Student’s t test).

Fig. 2.

Arid5a stabilizes IL-6 mRNA by recognizing the IL-6 3′ UTR. (A) Schematic diagram of expression constructs of Arid5a WT and mutant. The Arid is filled at black, and the amino acids present in the WT and mutant construct are indicated. (B–E and G) HEK293T cells were transfected with the pGL3-luciferase vector encoding the full length of the IL-6 3′ UTR, the fragments of the IL-6 3′ UTR (1–92, 1–142, 58–173, 122–197, 172–403), the full length of the TNF-α 3′ UTR or the full length of the IL-12 3′ UTR, or pGL3 empty vector, together with WT Arid5a expression vector (black bar; B, D, E, and G), ΔMutant Arid5a (gray bar; C), or empty vector. Luciferase activity was determined 24 h after transfection and normalized to that of pGL3-empty vector. The values were shown as relative to normalized after transfection with empty vector. (F) Schematic diagram of the full length of the IL-6 3′ UTR, and the fragments of the IL-6 3′ UTR. (H and I) HEK293 Tet-off cells were transfected with pTREtight-IL-6-CDS + 3′ UTR or pTREtight-IL-6-CDS vector, together with Arid5a expression vector or control (empty) vector. Cells were uniformly divided 3 h after transfection and incubated overnight. Total RNAs were prepared after dox (1 μg/mL) treatment, and IL-6 mRNA levels were determined by qPCR. The values were shown normalized to time 0 of dox addition (100%), respectively. (B–E and G–I) Data show means ± SD of three independent experiments. ***P < 0.001 (Student’s t test).

Fig. 3.

Arid5a deficiency (Arid5a^−/−) reduces IL-6 and TNF serum levels in LPS-treated mice. (A and B) WT and Arid5a-deficient (Arid5a^−/−) mice (6–8 wk old) were i.p. injected with LPS (10 mg/kg). Serum levels of IL-6 (A) or TNF-α (B) in WT and Arid5a^−/− mice were measured by ELISA at indicated time points after LPS challenge, respectively. Data show means ± SD of three independent experiments. ***P < 0.0001; **P < 0.001; *P < 0.05 (Student’s t test).

Fig. 4.

Arid5a deficiency (Arid5a^−/−) inhibits the development of EAE. (A) EAE disease score in WT and Arid5a^−/− mice (mean score ± SEM, n = 7, **P < 0.01). (B) IL-6 and TNF-α serum samples in WT and Arid5a^−/− mice were prepared at 20 d after immunization. Levels of IL-6 and TNF-α serum were measured by ELISA; n = 5, ***P < 0.001 (Student’s t test). (C–E) Frequencies of IL-17, IFN-γ, and Foxp3-positive CD4⁺ T cells in inguinal lymph node cells isolated from mice immunized with MOG_35–55. Numbers above bracketed lines indicate percent CD4⁺IL-17⁺, CD4⁺IFN-γ⁺, and CD4⁺Foxp3⁺ T cells. (F) Inguinal lymph node cells after immunization were restimulated with MOG_35–55 peptide (50 μg/mL) for 72 h. Secretions of IL-17 and IFN-γ in the supernatants were measured by ELISA. (C–F) Data are representative of two independent experiments with three mice per group (means ± SD, as in F).

Fig. 5.

Arid5a inhibits the destabilizing function of Regnase-1 on IL-6 mRNA. (A) HEK293T cells were transfected with the pGL3-luciferase vector encoding the IL-6 3′ UTR (1–403) or pGL3 empty vector, together with empty vector, Arid5a expression vector, Regnase-1 expression vector, or Arid5a and Regnase-1 expression vectors. Luciferase activity was determined 24 h after transfection and normalized to that of pGL3-empty vector. The values were shown as relative to normalized after transfection with empty vector. (B) HEK293 Tet-off cells were transfected with pTREtight-IL6-CDS + 3′ UTR vector, together with Arid5a expression vector, Regnase-1 expression vector, control (empty) vector or Arid5a and Regnase-1 expression vectors. The cells were uniformly divided 3 h after transfection, and incubated overnight. Total RNAs were prepared at the indicated times after dox (1 μg/mL) treatment, and IL-6 mRNA levels were determined by qPCR. The values were shown normalized to time 0 of dox addition (100%), respectively. All data show means ± SD of three independent experiments.