A role for IL-10 in the transcriptional regulation of Roquin-1

Abstract

Roquin-1, a RING finger E3 ubiquitin ligase, functions as a modulator of inflammation; however, nothing is known about how Rc3h1 expression is regulated. Here, we describe an opposing relationship between Roquin-1 and the IL-17 proinflammatory cytokine by demonstrating that enforced expression of Rc3h1 restricts Il17a expression, and that exposure of T cells to IL-10, a cytokine with immunosuppressive activity, increases Rc3h1 expression. Luciferase reporter assays conducted using eight transcription factor plasmids (STAT1, STAT3, STAT5, GATA2, c-Rel, IKZF1, IKZF2, and IKZF3) demonstrated that STAT1, STAT3, GATA2, and c-Rel increased Rc3h1 promoter activity, whereas IKZF2 decreased activity. Gene expression of those five transcription factors increased in T cells exposed to IL-10. Transcription factor-specific siRNAs suppressed the IL-10 effect on Rc3h1 transcription. These findings identify a role for IL-10 in regulating Rc3h1 transcription, and they have implications for understanding how Roquin-1 controls the immune response.

Keywords: Cytokine; Gene expression; IL-10; Luciferase; Promoter; Transcription factor; siRNA.

Fig. 1

Opposing relationship between Roquin-1 and IL-17. (A) MLN T cells 48 hr after stimulation using the Th17 induction regimen described in the Materials and Methods. (B) MLN T cells 48 hr after CD3 stimulation in the absence of Th17 induction. Cells were gated onto the CD4⁺ cell population. Representative histograms from three experiments. (C, D) Increased Il17a and decreased Rc3h1 transcription in MLN T cells following 24 and 48 hr of Th17 induction. Control cultures consisted of plate-bound anti-hamster antibody with normal hamster IgG. (E) Increased Rc3h1 transcription in EL4 cells following exposure to IL-10. (F) Exposure of EL4 cells to IL-10 resulted in increased Socs3 transcription. (G) Western blot showing increased levels of Roquin-1 protein expression in EL4 cells transfected with the Roquin-1 pEF6 expression plasmid. Ponceau S staining was done to compare sample protein levels. (H, I) Roquin-1 pEF6 transfection of EL4 cells resulted in increased Rc3h1 transcription and decreased Il17a transcription. Data are mean values ± SEM of at least three experiments.

Fig. 2

Transcriptional regulation of Rc3h1. (A) A web-based search for transcriptional factors that bind to the Rc3h1 promoter identified binding sites for STATs, GATA, IKZF and c-Rel. Luciferase reporter assays were used to evaluate the activational effects of eight transcription factors on the Rc3h1 promoter regions. Using a (B) -2.1 kb Rc3h1 promoter region, STAT1, STAT3, GATA2, and c-Rel resulted in a significant increase in luciferase activity; IKZF2 resulted in a significant decrease. STAT1, STAT3, GATA2, and c-Rel also resulted in increased luciferase activity using the (C) -1.3 kb and (D) -0.2 kb Rc3h1 promoter regions. Values are fold induction of luciferase activity of transcription factor-transfected cells relative to non-transcription factor transfected cells. Data are mean values ± SEM of 3-4 independent experiments.

Fig. 3

ChIP assays showing GATA2 interactions with: (A) -2.2 kb, (B) -1.8 kb, (C) -1.0 kb, and (D) -0.4 kb RC3H1 promoter regions. Data in each panel represent one ChIP experiment; the average values of all four data entries for the GATA2 ChIP assays (anti-GATA2 antibody) was significantly greater (p<0.001) than that of ChIP assay results done with None (mock) transfections, or STAT1 transcription factor transfections. (E, F) ChIP assays showing the effect of IL-10 on transcription factor interactions with the Rc3h1 promoter.

Fig. 4

IL-10 increased Rc3h1 transcription factor expression. (A) EL4 cells were cultured in the presence of mouse rIL-10, or rTGFβ as a control cytokine. Gene expression was measured at 0, 24, and 48 hr. Compared to the rTGFβ control cytokine, exposure of cells to IL-10 resulted in a significant increase in gene expression of STAT1, STAT3, GATA2, and c-Rel, as well as a non-statistical increase in IKZF2 48 hr post-IL-10 treatment. Data are mean values ± SEM of 3 experiments. (B) EL4 cells were transfected with siRNAs to either STAT1, STAT3, GATA2, c-Rel, IKZF2, a scramble control siRNA, or no siRNA for 4 hr followed by exposure to rIL-10 as described in the Materials and Methods. Cells were harvested and Rc3h1 expression was measured by qRT-PCR. All transcription factor-specific siRNAs decreased Rc3h1 expression relative to the control siRNA-transfected cells. For STAT1 and STAT3, this occurred in a statistically-significant manner. (C) Efficiency of knockdown of transcription factor expression. Data are mean values ± SEM of 3 experiments. ** p<0.01, * p<0.05, ◆ p<0.1 and >0.05. (D) A model whereby IL-10 exerts an effect on the transcriptional regulation of Rc3h1.