MicroRNA-21 regulates T-cell apoptosis by directly targeting the tumor suppressor gene Tipe2

Abstract

MicroRNAs (MiRs) are short noncoding RNAs that can regulate gene expression. It has been reported that miR-21 suppresses apoptosis in activated T cells, but the molecular mechanism remains undefined. Tumor suppressor Tipe2 (or tumor necrosis factor-α-induced protein 8 (TNFAIP8)-like 2 (TNFAIP8L2)) is a newly identified anti-inflammatory protein of the TNFAIP8 family that is essential for maintaining immune homeostasis. We report here that miR-21 is a direct target of nuclear factor-κB and could regulate Tipe2 expression in a Tipe2 coding region-dependent manner. In activated T cells and macrophages, Tipe2 expression was markedly downregulated, whereas miR-21 expression was upregulated. Importantly, Tipe2-deficient T cells were significantly less sensitive to apoptosis. Conversely, overexpression of Tipe2 in EL-4 T cells increased their susceptibility to activation-induced apoptosis. Therefore, Tipe2 provides a molecular bridge between miR-21 and cell apoptosis; miR-21 suppresses apoptosis in activated T cells at least in part through directly targeting tumor suppressor gene Tipe2.

Figure 1

NF-κB-dependent downregulation of Tipe2 in T cells and myeloid cells. (a) Murine naive CD4+ T cells were either untreated or treated with plate-bound anti-CD3 (1 μg/ml) and soluble anti-CD28 (1 μg/ml) for the indicated times, and Tipe2 mRNA levels were determined by quantitative PCR. (b) Murine naive CD4+ T cells were either untreated or treated with plate-bound anti-CD3 (1 μg/ml) and soluble anti-CD28 (1 μg/ml) for 5 h, Tipe2 protein levels were determined by western blot analysis. (c) Murine naive CD4+ T cells were treated as in (a) for 4 h in the absence or presence of NF-κB inhibitor Bay 11-7082. Tipe2 mRNA levels were determined by quantitative PCR. *P<0.02. The results are representative of three independent experiments

Figure 2

NF-κB binds to and activates the miR-21 promoter. (a) Murine naive CD4+ T cells were either untreated or treated with plate-bound anti-CD3 (1 μg/ml) and soluble anti-CD28 (1 μg/ml) for 6 h in the absence or presence of NF-κB inhibitor Bay 117-82, and miR-21 levels were determined by quantitative PCR. *P<0.01. (b) Murine bone marrow-derived macrophages were either untreated or treated with LPS (100 ng/ml) for 4 h in the absence or presence of NF-κB inhibitor Bay 117-82. MiR-21 levels were determined by quantitative PCR. *P<0.02. (c) RAW264.7 cells were transiently transfected with WT or NF-κB site-mutated murine miR-21 promoter luciferase construct. After 24 h, cells were either untreated or treated with LPS (100 ng/ml) for 6 h, and the luciferase activities measured. The promoter activity is presented as fold increase over untreated cells transfected with NF-κB site no. 2 mutated construct. To normalize the transfection efficiency across samples, the Renilla luciferase expression vector pRL-TK was included as an internal control. *P<0.01. (d) Nuclear extracts were prepared from mouse bone marrow-derived macrophages after stimulation for 2 h with LPS. Biotinylated miR-21 oligonucleotides containing NF-κB binding site nos. 1 or 2 or their mutants were absorbed by streptavidin–agarose beads, and then added to the nuclear extracts. The amount of p65 proteins in the precipitates were assessed by immunoblotting with anti-p65. (e) Mouse bone marrow-derived macrophages were treated with LPS (100 ng/ml). After 2 h, cells were fixed, and ChIP was performed using anti-p65, anti-RNA polymerase II (PolII), or control IgG. Immunoprecipitated DNA was analyzed by PCR using primers for NF-κB site on miR-21 promoter. Data are representative of two independent experiments

Figure 3

Tipe2 coding region, but not 3′-UTR, is responsible for the downregulation of Tipe2. EL4 cells were transiently transfected with murine Tipe2 promoter luciferase construct with Tipe2 3′-UTR (A) or coding region (+307 to +644) (a and b) inserted downstream of the luciferase gene but upstream of polyA signal sequence. For (b), expression vector for full-length p65, or the empty vector as indicated, was co-transfected. At 24 h after transfection, cells were treated with phorbol myristyl acetate (PMA) (50 ng/ml) and ionomycin (1 μM) for 5 h, and the luciferase activities measured. The promoter activity is presented as fold increase over cells transfected with murine miR-21 promoter only luciferase construct (a) or empty vector (b). To normalize the transfection efficiency across samples, the Renilla luciferase expression vector pRLTK was included as an internal control. *P<0.01. Data are representative of three independent experiments

Figure 4

MiR-21 targets Tipe2 in a coding region-dependent manner. (a) Deletion mutants of Tipe2 coding region (+307 to +644) were analyzed in the luciferase reporter assay. Putative binding site for miR-21, miR-155 and miR-23b are indicated. (b) The predicted miR-21 binding site on murine Tipe2 coding region and the mutation strategy is shown. The ‘X' indicates the mutated sites. (c) WT and miR-21 binding site-mutated Tipe2 coding region constructs were analyzed in the luciferase reporter assay. The ‘X' indicates the mutated sites. *P<0.02. Data are representative of three independent experiments

Figure 5

MiR-21 regulates endogenous Tipe2 expression. RAW264.7 (a) and EL4 (b) cells were transfected with miR-21 mimic or negative control as indicated. At 24 h after transfection, miR-21, Tipe2 and Pdcd4 mRNA levels were determined by real-time RT-PCR. *P<0.02. Data are representative of three independent experiments

Figure 6

Tipe2 deficiency in T cells renders them resistant to death and miR-21 regulates T-cell apoptosis through Tipe2. (a) Splenic naive CD4+ T cells were isolated from WT and Tipe2^−/− mice (n=3) and either untreated or treated with plate-bound anti-CD3 (1 μg/ml) and soluble anti-CD28 (1 μg/ml) for the indicated times. Cells were then stained with annexin V and 7AAD, and the degree of apoptosis was analyzed by flow cytometry. (b) EL4 cells were transfected with miR-21 mimic or negative control together with Tipe2-overexpressing construct or an empty vector. After 24 h, cells were either untreated or treated with plate-bound anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml). After 48 h, cells were stained with annexin V and 7AAD, and the degree of apoptosis was analyzed by flow cytometry. *P<0.01. Data are representative of three independent experiments

Figure 7

NF-κB inhibits activation-induced T-cell apoptosis through the miR-21–Tipe2 axis. TCR and anti-CD28 stimulation activate NF-κB through IKK (by degrading the inhibitor protein IκB). After activation, NF-κB translocates into the nucleus and turns on its target genes, including miR-21. MiR-21 in turn degrades Tipe2 and inhibits activation-induced T-cell apoptosis