Overexpression of the Transcription Factor Sp1 Activates the OAS-RNAse L-RIG-I Pathway

<div><p>Deregulated expression of oncogenes or transcription factors such as specificity protein 1 (Sp1) is observed in many human cancers and plays a role in tumor maintenance. Paradoxically in untransformed cells, Sp1 overexpression induces late apoptosis but the early intrinsic response is poorly characterized. In the present work, we studied increased Sp1 level consequences in untransformed cells and showed that it turns on an early innate immune transcriptome. Sp1 overexpression does not activate known cellular stress pathways such as DNA damage response or endoplasmic reticulum stress, but induces the activation of the OAS-RNase L pathway and the generation of small self-RNAs, leading to the upregulation of genes of the antiviral RIG-I pathway at the transcriptional and translational levels. Finally, Sp1-induced intrinsic innate immune response leads to the production of the chemokine CXCL4 and to the recruitment of inflammatory cells <i>in vitro</i> and <i>in vivo</i>. Altogether our results showed that increased Sp1 level in untransformed cells constitutes a novel danger signal sensed by the OAS-RNase L axis leading to the activation of the RIG-I pathway. These results suggested that the OAS-RNase L-RIG-I pathway may be activated in sterile condition in absence of pathogen.</p></div

A schematic overview of the signaling pathway triggered by increased Sp1 level.

<p>Upon Sp1 overexpression, <i>Oas2</i> and <i>Rnasel</i> genes are upregulated and small self-RNAs are produced. Sensing of small self-RNAs leads to the activation of the sensor RIG-I, the IRF3/7 transcription factors and downstream effector targets such as ISGs.</p

Sp1 activates the OAS-RNAse L pathway and the production of small self-RNAs.

<p>(A) BaF3-Sp1 inducible cell line was grown with (white bars) or without (black bars) dox for the indicated times, and relative mRNA levels of OAS2 and RNase L genes were quantified by RT-qPCR. (B, D, E) 3T3 (B) and MEFs (D, E) WT or deficient for the RNase L (RNase L KO) or MAVS (MAVS KO) were transduced with Sp1, Sp1 Zn^2,3 (Zn) or empty vector (EV). Transduced cells (CD2 positive) were purified 72 h post-transduction by magnetic selection and relative mRNA levels of indicated genes were quantified by RT-qPCR. (C) Small RNAs from BaF3-Sp1 cells following induction Sp1 or not were extracted at the indicated times, and transfected into 293HEK cells (5μg per condition). Luciferase reporter assay was performed to analyze the ISRE promoter activity. Data are mean values ± standard deviation (SD) from one experiment representative of two or three independent experiments. Statistical analysis were performed using 2-tailed <i>t</i> tests. Levels of significance are expressed as follows: *<i>P</i> <0.05; **<i>P</i> <0.01.</p

Functional analysis of Sp1 signature.

<p>The gene list enrichment analysis from the Gene Ontology of the SP1 specific signature was performed with g:Profiler. The moderate hierarchical filtering used here allows a compact representation of gene list enrichment results. Significantly enriched GO terms containing less than 5 genes were excluded.</p><p>Functional analysis of Sp1 signature.</p

Sp1 overexpression activates genes of the RIG-I pathway.

<p>(A-B) BaF3 (A) and 3T3 (B) cells were transduced with retroviruses encoding wild-type Sp1, Sp1 Zn^2,3 mutant (Zn) that does not bind DNA, or empty vector (EV). Transduced cells (CD2 positive) were purified by magnetic selection 30 h (BaF3) or 72 h (3T3) later, and mRNA levels of indicated genes were measured by RT-qPCR (TLDA used in B) and normalized to housekeeping genes (HPRT in A and Rps-21 in B) mRNA levels. Data are representative of one out of three independent experiments in A. In B panel, data are representative of two independent experiments and statistical analysis was performed using 2-tailed <i>t</i> tests. Levels of significance are expressed as follows: **<i>P</i> <0.01. (C) BaF3-Sp1 inducible cell line was grown with (white bars) or without (black bars) dox for indicated times, and relative mRNA levels of indicated genes were quantified by RT-qPCR (TLDA). Data are representative of one out of four independent experiments.</p

Proteins of the antiviral RIG-I pathway are induced by increased Sp1 level.

<p>BaF3-Sp1 cells were grown in the presence or absence of dox. (A) Cell extracts were collected at indicated time and expression of the indicated proteins was analyzed by immunoblot. A549 cells treated 16 h with IFNa (1000U/ml) were used as positive control (IFN) for MDA5 and RIG-I induction. (B) BaF3-Sp1 cells were cultured for 20 h, fixed and stained for RIG-I (red) and analyzed by fluorescent microscopy. The percentage of cells presenting RIG-I punctuated staining is indicated, and corresponds to the mean of 8 independent acquisitions. Data are representative of one out of two independent experiments.</p

Immune cells are recruited by cells overexpressing Sp1.

<p>(A) BaF3-Sp1 cells were grown in the presence (+) or absence (-) of dox. CXCL4 production by BaF3-Sp1 was measured at the indicated times by ELISA. (B) BaF3-Sp1 cells were treated with or without dox for 24 h and incubated in the presence of neutrophils into transwell chambers during 3 h. Neutrophils recruitment (Ly6G⁺ CD11b⁺ cells) was assessed by flow cytometry. (C) BaF3 and BaF3-Sp1 cells were grown 18 h in absence or presence of dox and injected intra-peritoneally into mice. PBS injected mice were used as controls (Naive). Peritoneal washes were performed 6 h after injection. Cells were then harvested and cellular recruitment (CD11b⁺ Ly6G^- cells) was estimated by flow cytometry. These results are from one representative out of three independent experiments. Statistical analysis were performed using 2-tailed <i>t</i> tests. Levels of significance are expressed as follows: *<i>P</i> <0.05; **<i>P</i> <0.01; ***<i>P</i> = 0.0001; ****<i>P</i> <0.0001.</p