Role of a transductional-transcriptional processor complex involving MyD88 and IRF-7 in Toll-like receptor signaling - PubMed (original) (raw)

Role of a transductional-transcriptional processor complex involving MyD88 and IRF-7 in Toll-like receptor signaling

Kenya Honda et al. Proc Natl Acad Sci U S A. 2004.

Abstract

Toll-like receptor (TLR) activation is central to immunity, wherein the activation of the TLR9 subfamily members TLR9 and TLR7 results in the robust induction of type I IFNs (IFN-alpha/beta) by means of the MyD88 adaptor protein. However, it remains unknown how the TLR signal "input" can be processed through MyD88 to "output" the induction of the IFN genes. Here, we demonstrate that the transcription factor IRF-7 interacts with MyD88 to form a complex in the cytoplasm. We provide evidence that this complex also involves IRAK4 and TRAF6 and provides the foundation for the TLR9-dependent activation of the IFN genes. The complex defined in this study represents an example of how the coupling of the signaling adaptor and effector kinase molecules together with the transcription factor regulate the processing of an extracellular signal to evoke its versatile downstream transcriptional events in a cell. Thus, we propose that this molecular complex may function as a cytoplasmic transductional-transcriptional processor.

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Figures

Fig. 1.

Fig. 1.

Subcellular localization of IRFs and MyD88. (a) Confocal images of HEK293T cells transiently expressing IRF-3YFP, IRF-7YFP, MyD88CFP, or TRIFCFP. (b) Confocal images of HEK293T cells coexpressing MyD88CFP with IRF-3YFP or IRF-7YFP. (c) HeLa cells were transfected with expression plasmids for IRF-7YFP or IRF-3YFP and MyD88CFP. FRET, YFP, and CFP images were collected by using an inverted fluorescence microscope equipped with a cooled charge-coupled device camera. FRETC was determined as described in the text and presented in the pseudocolor mode as indicated below the photographs.

Fig. 2.

Fig. 2.

Nuclear translocation of IRF-7 induced by CpG-A ODN stimulation. HeLa cells expressing IRF-7YFP alone (a), MyD88CFP and IRF-7YFP (b), or MyD88CFP and IRF-3YFP (c) were placed on a time-lapse microscope and imaged every minute. Cells were stimulated with 1 μM CpG-A plus DOTAP and incubated for up to 2 h. Fluorescent images of YFP at the indicated periods are shown. CFP images indicate the expression level of MyD88. N, nucleus.

Fig. 3.

Fig. 3.

Association of IRF-7 with MyD88 and TRAF6. (a) Coimmunoprecipitation of IRF-7 with MyD88. HEK293T cells were transfected transiently with various concentrations of pEF-HA-IRF-7 (0, 0.17, 0.5, and 2.0 μg) together with pCXN2-FLAG-MyD88 (2.0 μg) in six-well plates, and cell lysates were subjected to immunoprecipitation with the anti-FLAG antibody. IRF-7 was immunoblotted with a rabbit polyclonal anti-HA antibody. MyD88 was immunoblotted with anti-FLAG antibody. (b) HEK293T cells were transfected transiently with the indicated combinations of pEF-HA-IRF-7 (2.0 μg) and pME-FLAG-TRAF6 (2.0 μg), and cell lysates were subjected to immunoprecipitation with the anti-FLAG antibody. (c) Schematic structures of the deletion mutants of MyD88. (d) HEK293T cells were transfected transiently with the indicated combinations of HA-tagged IRF-7 (2.0 μg) and FLAG-tagged full-length or deletion mutants of MyD88 (2.0 μg) and subjected to immunoprecipitation with the anti-FLAG antibody.

Fig. 4.

Fig. 4.

IRF-7 activation by MyD88 and TRAF6. HEK293T cells were cotransfected transiently with p125-Luc (50 ng) and the expression vector for the indicated combinations of MyD88 (10 ng), MyD88 mutants (10 ng), IRF-3 (0, 1, or 10 ng), IRF-7 (10 ng in b; 0, 1, or 10 ng in a and d), and TRAF6 [10 ng in b; 0, 1, 5 or 10 ng in _c_]. After 24 h of transfection, cells were harvested, and luciferase activity was measured.

Fig. 5.

Fig. 5.

Involvement of IRAK4 in the TLR9/7-dependent induction of IFN-α in pDCs. (a) The effect of IRF-7 expression on MyD88- or TRAF6-mediated NF-κB activation was monitored by using NF-κB-luc. HEK293T cells were cotransfected transiently with NF-κB-luc (50 ng) and the expression vectors for the indicated combinations of full-length MyD88, mutant MyD88 (10 ng), TRAF6 (100 ng), and IRF-7 (0, 1, 5, or 10 ng). Luciferase activity was measured 24 h after transfection. (b) IFN-α induction in pDCs stimulated by CpG-A or single-stranded RNA depends on MyD88 and IRAK4. Splenic pDCs (B220+CD11cint cells) were sorted and stimulated with 3 μM CpG-A ODN or 5 μg/ml poly(uridylic acid) plus DOTAP for 24 h. The concentration of IFN-α in culture supernatants was measured by ELISA. (c) Schematic illustration of the CTTP, providing a model of the TLR-dependent MyD88 signaling that potentially processes the signal to activate distinct transcription factors by means of the identified CTTP complex. For consistency with a previous report (19-21), IRAK1 also is included, although this molecule was not examined in this study. The complex may be in dynamic state depending on the expression levels of these molecules in this complex, as demonstrated in a. In normally growing cells, in which the IRF-7 level is very low, the CTTP complex may be shifted to the NF-κB pathway, but it may change in favor of the IRF-7 pathway when its expression level increases, owing to the capability of IRF-7 to interact with both MyD88 and TRAF6. Additionally, the CTTP complex may be regulated by their distinct compartmentalization in a cell. Although this model is consistent with our present data, it needs to be clarified further in the physiological context, for example, by examining DCs lacking IRF-7.

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