IRF-3-dependent, NFkappa B- and JNK-independent activation of the 561 and IFN-beta genes in response to double-stranded RNA - PubMed (original) (raw)

IRF-3-dependent, NFkappa B- and JNK-independent activation of the 561 and IFN-beta genes in response to double-stranded RNA

Kristi L Peters et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Double-stranded (ds) RNA induces transcription of the 561 gene by activating IFN regulatory factor (IRF) transcription factors, whereas similar induction of the IFN-beta gene is thought to require additional activation of NFkappaB and AP-1. In mutant P2.1 cells, dsRNA failed to activate NFkappaB, IRF-3, p38, or c-Jun N-terminal kinase, and transcription of neither 561 mRNA nor IFN-beta mRNA was induced. The defect in the IRF-3 pathway was traced to a low cellular level of this protein because of its higher rate of degradation in P2.1 cells. As anticipated, in several clonal derivatives of P2.1 cells expressing different levels of transfected IRF-3, activation of IRF-3 and induction of 561 mRNA by dsRNA was restored fully, although the defects in other responses to dsRNA persisted. Surprisingly, IFN-beta mRNA also was induced strongly in these cells in response to dsRNA, demonstrating that the activation of NFkappaB and AP-1 is not required. This conclusion was confirmed in wild-type cells overexpressing IRF-3 by blocking NFkappaB activation with the IkappaB superrepressor and AP-1 activation with a p38 inhibitor. Therefore, IRF-3 activation by dsRNA is sufficient to induce the transcription of genes with simple promoters such as 561 as well as complex promoters such as IFN-beta.

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Figures

Figure 1

P2.1 cells display multiple dsRNA-mediated signaling defects. (A) Kinetics of degradation of IκBα in U4C cells. U4C cells were treated with dsRNA for the times indicated. Cell lysates were prepared, and Western analysis was performed with an antibody against IκBα. (B) Lack of IκBα degradation in P2.1 cells. U4C and P2.1 cells were left untreated (−) or treated with dsRNA for 1 h (+). Cell lysates were prepared, and Western analysis for IκBα was performed as described for A. (C) Failure of dsRNA-mediated IRF-3 translocation in P2.1 cells. U4C and P2.1 cells were left untreated or treated with dsRNA for 1 h. The cells were fixed, and immunofluorescence was performed to detect the nuclear translocation of IRF-3 in response to dsRNA. (D) Lack of induction of 561 mRNA in response to dsRNA in P2.1 cells. U4C and P2.1 cells were left untreated (−) or treated with dsRNA for 6 h (+). RNA was harvested, and an RPA was performed to detect the induction of 561 message. Actin mRNA served as an internal control. (E) Failure of p38 activation by dsRNA in P2.1 cells. U4C and P2.1 cells were treated with dsRNA for the times indicated. Cell lysates were prepared, and Western analyses were performed with antibodies against p38 or activated, phosphorylated p38 (P-p38). (F) Lack of JNK activation by dsRNA in U4C and P2.1 cells. U4C and P2.1 cells were treated with dsRNA for the times indicated. Cell lysates were prepared, and Western analyses were performed with antibodies against JNK or activated, phosphorylated JNK (P-JNK).

Figure 2

The level of IRF-3 protein but not mRNA is lower in mutant P2.1 cells. (A) The IRF-3 and actin protein levels in U4C and P2.1 cells were determined by Western analysis. The relative levels of IRF-3 in U4C and P2.1 cells, normalized against the actin levels, are shown beneath the figure. (B) IRF-3 and actin mRNA levels were determined by using RPA and quantified by using a Molecular Dynamics PhosphorImager. The relative normalized levels of IRF-3 mRNA are denoted beneath the figure.

Figure 3

The degradation rate of IRF-3 is increased in P2.1 cells. (A) Establishment of U4C and P2.1 cell lines expressing IRF-3. P2.1 and U4C cells were transfected with an IRF-3 expression plasmid, and individual clones were isolated. Western analysis was performed to determine IRF-3 and actin expression levels. The normalized IRF-3 expression levels relative to that in U4C cells are given below the lanes. (B) Determination of IRF-3 half-life. U4C.2 (■) and P2.1.17 (□) cells were pulse-labeled with a mixture of 35S-labeled methionine and cysteine for 2 h. The label then was chased for the indicated times, and cell lysates were prepared. IRF-3 was immunoprecipitated from equal amounts of whole-cell extract and separated by SDS/PAGE. Radiolabeled IRF-3 was quantified with a Molecular Dynamics PhosphorImager.

Figure 4

Expression of IRF-3 in P2.1 cells restores IRF-3 activation and 561 induction. (A) Restoration of dsRNA-induced activation of IRF-3. P2.1.6 cells were treated with dsRNA and stained for IRF-3 immunofluorescence as described for Fig. 1_C_. (B) Restoration of 561 mRNA induction by dsRNA in IRF-3-expressing cells. Cells were left untreated (−) or treated with dsRNA for 6 h (+) and examined for the induction of 561 mRNA by RPA as described for Fig. 1_D_. The normalized 561 mRNA level in treated U4C cells was set to 10. The results of three separate experiments were averaged, and the graph represents the mean ± SD.

Figure 5

Expression of IRF-3 does not restore other signaling defects of P2.1 cells but restores IFN-β mRNA induction. (A) dsRNA-induced activation of NFκB. U4C, P2.1, and the P2.1 clones expressing IRF-3 cells were left untreated (−) or treated with dsRNA for 60 min (+). An EMSA for NFκB was performed. (B) Lack of p38 activation. P2.1.17 cells were treated with dsRNA for the indicated times or anisomycin (An) for 15 min. Activation of p38 was determined by Western analysis as described for Fig. 1_E_. (C) Induction of IFN-β mRNA by dsRNA treatment. U4C, P2.1, and P2.1.17 cells were treated with dsRNA for 6 h (+) or left untreated (−). RNA was harvested, and an RPA was performed with probes for IFN-β and actin mRNAs.

Figure 6

NFκB activation is not required for the induction of IFN-β mRNA by dsRNA. (A) Western analysis of IκBαSR, IRF-3, and actin expression. 2fTGH cell lines expressing the IκBαSR and IRF-3 were established as described in Materials and Methods. Western analyses of the clones were performed with appropriate antibodies. (B) NFκB activation in response to dsRNA treatment. Cells were treated with dsRNA for 1 h (+) or left untreated (−). EMSA analysis of cell extracts was performed with the NFκB probe. (C) Induction of IFN-β mRNA after dsRNA treatment. Cells were treated with dsRNA for 6 h (+) or left untreated (−). RNA was harvested, and an RPA was performed with probes against IFN-β and actin mRNAs.

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IRF-3-dependent, NFkappa B- and JNK-independent activation of the 561 and IFN-beta genes in response to double-stranded RNA - PubMed (original) (raw)