BCL11B enhances TCR/CD28-triggered NF-kappaB activation through up-regulation of Cot kinase gene expression in T-lymphocytes

Abstract

BCL11B is a transcriptional regulator with an important role in T-cell development and leukaemogenesis. We demonstrated recently that BCL11B controls expression from the IL (interleukin)-2 promoter through direct binding to the US1 (upstream site 1). In the present study, we provide evidence that BCL11B also participates in the activation of IL-2 gene expression by enhancing NF-kappaB (nuclear factor kappaB) activity in the context of TCR (T-cell receptor)/CD28-triggered T-cell activation. Enhanced NF-kappaB activation is not a consequence of BCL11B binding to the NF-kappaB response elements or association with the NF-kappaB-DNA complexes, but rather the result of higher translocation of NF-kappaB to the nucleus caused by enhanced degradation of IkappaB (inhibitor of NF-kappaB). The enhanced IkappaB degradation in cells with increased levels of BCL11B was specific for T-cells activated through the TCR, but not for cells activated through TNFalpha (tumour necrosis factor alpha) or UV light, and was caused by increased activity of IkappaB kinase, as indicated by its increase in phosphorylation. As BCL11B is a transcription factor, we investigated whether the expression of genes upstream of IkappaB kinase in the TCR/CD28 signalling pathway was affected by increased BCL11B expression, and found that Cot (cancer Osaka thyroid oncogene) kinase mRNA levels were elevated. Cot kinase is known to promote enhanced IkappaB kinase activity, which results in the phosphorylation and degradation of IkappaB and activation of NF-kappaB. The implied involvement of Cot kinase in BCL11B-mediated NF-kappaB activation in response to TCR activation is supported by the fact that a Cot kinase dominant-negative mutant or Cot kinase siRNA (small interfering RNA) knockdown blocked BCL11B-mediated NF-kappaB activation. In support of our observations, in the present study we report that BCL11B enhances the expression of several other NF-kappaB target genes, in addition to IL-2. In addition, we provide evidence that BCL11B associates with intron 2 of the Cot kinase gene to regulate its expression.

Figure 1. BCL11B regulates expression of IL-2 through site(s) located between −210 and −190 in addition to the US1 site

(A) Schematic representation of the IL-2 promoter. (B) Ratios of Luciferase reporter assays between MSCV-BCL11B and MSCV Jurkat cells transfected with the IL-2 promoter deletion mutants. Reporter assays were conducted after treatment of the cells with PMA/ionomycin for 8 hours. The quantification represents the means of three independent experiments ± SD.

Figure 2. BCL11B activates transcription from NFKB sites, but not from AP1 or NFAT

MSCV (open bars) and MSCV-BCL11B (solid bars) Jurkat cells were transfected with pNFkB-Luc(A), AP1-Luciferase or NFAT-luciferase (B), or 4xRE/AP1-Luciferase (C and D) and Renilla Luciferase plasmids. 24 hrs posttransfection, the cells were stimulated with 50 ng/ml PMA plus 1 μM Ionomycin for 6 hrs (A, B and C), or crosslinked anti-CD3 antibody (OKT3) (10 μg/ml) or anti-CD3 antibody plus soluble anti-CD28 antibody (2 μg/ml) for 10 hrs (D). Renilla Luciferase activity was used for normalization.

Figure 3. Endogenous BCL11B is required for full activation of transcription from the NF-kB site

Jurkat cells were transfected with 4xRE/AP1-Luciferase plasmid and the nontargeting siRNA (open bars) or BCL11B specific (solid bars) siRNAs. (A) Western blot analysis of BCL11B after transfection of Jurkat cells with BCL11B specific or nontargeting siRNAs. (B) Luciferase activity was measured in cells treated or not with 50 ng/ml PMA and 1 μM Ionomycin for 7 hrs.

Figure 4. BCL11B enhances nuclear levels of p50 and p65, as well as binding of NF-kB consensus sites following T cell activation

(A) EMSA using a labeled consensus NF-kB oligonucleotides and nuclear extracts prepared from Jurkat cells stimulated (lanes 2 to 8) or not (lane 1) with 50 ng/ml PMA and 1 μM Ionomycin for 1 hr. For the supershift experiments the nuclear extracts were incubated with the indicated antibodies for 15 min prior to incubation with the labeled oligonucleotide probe. (B) MSCV and MSCV-BCL11B Jurkat cells were stimulated or not with 50 ng/ml PMA and 1 μM Ionomycin for 1 hr (left panel), or with anti-CD3 and anti-CD28 (right panel). The nuclear extracts were analyzed by immunoblotting for p65 and p50 proteins. Actin was used as a loading control. Numbers indicate fold increase in the levels of p50 and p65 after normalization to actin, evaluated through densitometry. (C) EMSA using labeled consensus NF-kB oligonucleotides and nuclear extracts prepared from MSCV (lanes 1 through 4) and MSCV-BCL11B (lanes 5 through 8) Jurkat cells stimulated (lanes 2 to 4 and 6 to 8) or not (lanes 1 and 5) with 50 ng/ml PMA and 1 μM Ionomycin for 1 hr. For the supershift experiments the nuclear extracts were incubated with the indicated antibodies for 15 min prior to incubation with the labeled probe. NFkB-DNA complexes are indicated by the black arrow and the supershifted complexes by the gray arrow. Enhancement of binding as a result of BCL11B overexpression is indicated by asterisks.

Figure 5. BCL11B enhances IkB phosphorylation and degradation, and IkB kinase phosphorylation in a TCR-dependent manner

(A) MSCV and MSCV-BCL11B Jurkat cells were treated with 50 ng/ml PMA and 1 μM ionomycin for the indicated time points. IkB degradation was analyzed by Western blot analysis of cytoplasmic fractions with specific antibodies. Actin was used as loading control. (B) Same as in (a) except that the cells were irradiated with 80 J/m². (C) MSCV and MSCV-BCL11B Jurkat cells were pretreated with 2.5 μM MG132 for 16 hrs followed by stimulation with 50 ng/ml PMA and 1 μM ionomycin for the indicated time points and P-IkBα was recognized with specific antibodies. The same membrane was stripped and total amount of IkBα was detected by reprobing with an anti-IkBα antibody. (D) MSCV and MSCV-BCL11B Jurkat cells were treated with 50 ng/ml PMA and 1 μM Ionomycin for the indicated time points. The P-IKKα/β were detected with specific antibodies. The total amount of IKKα/β proteins is shown on the same membrane. (E) MSCV and MSCV-BCL11B Jurkat cells were treated with 20 ng/ml TNFα for the indicated time points. IkB degradation was analyzed by Western blot analysis of cytoplasmic fractions with specific antibodies. Actin was used as loading control. (F) (Left panel) HeLa cells ectopically expressing BCL11B (solid bars) or not (open bars) were transfected with pNF-kB-Luc and Renilla Luciferase plasmids. 24 hrs posttransfection the cells were stimulated with 50 ng/ml PMA and 1 μM ionomycin for 6 hrs and the luciferase activity was evaluated. (Right panel) Western blot showing ectopic expression of BCL11B in Hela cells.

Figure 6. Enhanced activation of NF-kB by BCL11B is mediated through Cot kinase gene expression

(A) MSCV (open bars) and MSCV-BCL11B (solid bars) Jurkat cells were treated or not with 50 ng/ml PMA and 1 μM ionomycin for 4 hrs. Cot mRNA expression was assessed by quantitative RT-PCR. The relative abundance of Cot kinase mRNA was normalized against actin in each sample. (B) (upper panel) MSCV (open bars) and MSCV-BCL11B (solid bars) Jurkat cells were transfected with the Cot dominant negative (DN) mutant construct, or Cot kinase siRNA, as indicated, and 4xRE/AP1-Luciferase and Renilla luciferase plasmids. 24 hours posttransfection the cells were stimulated with 50 ng/ml PMA and 1 μM ionomycin for 7 hrs and luciferase activity was determined. (lower panel, left) Expression of the Cot kinase dominant negative (DN) mutant construct detected by Western blot analysis in MSCV and MSCV-BCL11B Jurkat cells. (lower panel, right) Reduction in Cot kinase mRNA levels as a result of transfection with Cot kinase siRNA. Relative Cot kinase mRNA levels were evaluated by quantitative RT-PCR, as previously described [6, 7]. (C) MSCV (open bars) and MSCV-BCL11B (solid bars) Jurkat cells were transfected with Cot kinase siRNA or nontargeting siRNA, followed by treatment with 50 ng/ml PMA and 1 μM ionomycin. IL-2 mRNA expression was assessed by quantitative RT-PCRs. The relative abundance of IL-2 mRNA was normalized against actin in each sample.

Figure 7. Endogenous BCL11B binds to Cot kinase gene intron 2

Chromatin immunoprecipitation of Jurkat cells, stimulated or not with PMA/ionomycin, with anti-BCL11B antibodies or IgG. Eluted DNA was analyzed by quantitative PCR using primers for Cot kinase gene promoter (−519−300 and −270−40) or intron 2 (i590-i280 and i300-i25).