Role for p53 in gene induction by double-stranded RNA

Abstract

Cross talk between p53 and interferon-regulated pathways is implicated in the induction of gene expression by biologic and genotoxic stresses. We demonstrate that the interferon-stimulated gene ISG15 is induced by p53 and that p53 is required for optimal gene induction by double-stranded RNA (dsRNA), but not interferon. Interestingly, virus induces ISG15 in the absence of p53, suggesting that virus and dsRNA employ distinct signaling pathways.

FIG. 1

p53 induces ISG15 expression. (A) ISG15 protein (50 μg/lane) in cell lysates from HeLa or HeLa-ts cells incubated for the indicated times at 32°C, or following treatment with 200 U of IFN-α (Hoffmann-LaRoche)/ml for 18 h, was analyzed by Western blotting. (B) ISG15 mRNA in total RNA (12 μg/lane) from HeLa-ts cells incubated for 6 h at 32 or 37°C in the presence or absence of 50 μg of cycloheximide (CHX)/ml was determined by Northern blotting.

FIG. 2

p53 is required for ISG15 induction by dsRNA, but not IFN. (A) WT (+/+) and KO (−/−) p53 MEFs were treated with 200 U of IFN-α or -β/ml or 100 U of IFN-γ (Lee Biomolecular)/ml for 18 h or with 50 μg of poly(I-C) (dsRNA; Sigma)/ml for 12 h, and ISG15 mRNA in 20 μg of total RNA/lane was measured by Northern blotting. (B) ISG15 mRNA induction by dsRNA in p53+/+ (HCT-116 40.16) or p53−/− (HCT-116 379.2) cells was measured by Northern blotting (12 μg/lane).

FIG. 3

Virus induction of ISGs in p53-positive and -null HCT-116 cells. Expression of ISG15 (4), ISG43 (24), IRF1 (30), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (14) mRNAs in 12 μg of total RNA/lane from p53+/+ (HCT-116 40.16) or p53−/− (HCT-116 379.2) cells was measured by Northern blotting. Hybridizations were performed sequentially, and the blot was stripped of probe between hybridizations. Virus infection is described in the text; IFN-α treatment was at 200 U/ml for 12 h; dsRNA treatment was for 12 h with 50 μg of poly(I-C)/ml.

FIG. 4

ISG15 promoter activity mimics endogenous ISG15 mRNA regulation by p53, dsRNA, and virus. Cells (6 × 10⁵ HCT 116) were seeded in 32-mm plates and allowed to attach overnight. Cells were transfected with 500 ng of pGL3/ISG15-Luc, 50 ng of pRL null (Promega), and 450 ng of pcDNA3 for carrier DNA by using Lipofectamine Plus (Life Technologies) following the manufacturer's instructions. Twenty-four hours posttransfection, the medium was aspirated and replaced with medium containing either 1,000 U of IFN-α/ml, 50 μg of dsRNA/ml, or Sendai virus (multiplicity of infection, 10). Cells were incubated for 12 h and then lysed, and luciferase assays were performed. Luciferase activity was assessed on 20 μl of each lysate as directed by the supplier (Dual Luciferase Kit, Promega) using a TD 20/20 luminometer (Turner Designs). Luciferase activity is presented as the ratio of firefly activity to renilla activity to control for differences in transfection efficiency. Each data point is the mean of triplicate samples ± the standard error; the data presented are representative of four independent experiments.