The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 179, 6867 -6872
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Funami, K.
Right arrow Articles by Matsumoto, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Funami, K.
Right arrow Articles by Matsumoto, M.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*Protein
*UniGene
*Substance via MeSH

Spatiotemporal Mobilization of Toll/IL-1 Receptor Domain-Containing Adaptor Molecule-1 in Response to dsRNA1

Kenji Funami2,*, Miwa Sasai3,*, Yusuke Ohba{dagger}, Hiroyuki Oshiumi*, Tsukasa Seya* and Misako Matsumoto4,*

* Department of Microbiology and Immunology, and {dagger} Department of Pathophysiology and Signal Transduction, Department of Pathology, Hokkaido University Graduate School of Medicine, Kita-ku Sapporo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
TLR3 recognizes viral dsRNA and induces antiviral immune responses. TLR3-mediated cell activation relies on Toll/IL-1R (TIR) domain-containing adaptor molecule-1 (TICAM-1, also named TIR domain-containing adaptor inducing IFN-β or TRIF), which recruits downstream signaling molecules to activate the transcription factors IFN regulatory factor 3 (IRF-3) and NF-{kappa}B. The mechanisms by which TICAM-1 is activated and transmits signals remain largely unknown. In this study we show that TICAM-1 alters its distribution profile from a diffuse cytoplasmic form to a speckle-like structure in response to dsRNA. The receptor-interacting protein 1 (RIP1), a crucial signaling molecule for TICAM-1-mediated NF-{kappa}B activation, accumulated in the TICAM-1 speckles. In addition, NF-{kappa}B-activating kinase-associated protein 1 (NAP1), a downstream molecule linking TICAM-1 and the IRF-3-activating kinase TBK1 (TANK-binding kinase 1), was also recruited to the TICAM-1 speckles. Notably, a transient colocalization of TICAM-1 and TLR3 was observed before the extensive formation of the TICAM-1 speckles. Thus, the spatiotemporal mobilization of TICAM-1 in response to dsRNA and the formation of the TICAM-1 speckles containing RIP1 and NAP1 are important for the activation of the TLR3-TICAM-1 pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The innate immune system acts as the first line of defense against numerous pathogens. Both membrane-bound and cytoplasmic receptors recognize pathogen-associated molecular patterns and induce a multiple array of antimicrobial immune responses (1). TLR3 and cytoplasmic retinoic acid-inducible gene I (RIG)-like receptors recognize viral dsRNA (2, 3, 4). TLR3 detects extracellular dsRNA, the mode being distinct from that of a cytoplasmic RIG-like receptor. TLR3-mediated signaling relies on the downstream adaptor molecule Toll/IL-1R (TIR)5 domain-containing adaptor molecule 1 (TICAM-1) (also called TIR domain-containing adaptor inducing IFN-β or TRIF), which activates the transcription factors IFN regulatory factor 3 (IRF-3), NF-{kappa}B, and MAPK, leading to the induction of type I IFN (especially IFN-β), cytokine/chemokine production, and dendritic cell (DC) maturation (5, 6).

Human TLR3 is predominantly expressed intracellularly in myeloid DCs and epithelial cells but not in plasmacytoid DCs (7, 8). TLR3 signaling arises in the intracellular compartment, but the mechanisms by which TICAM-1 transmits signals are still unclear. TICAM-1 possesses a proline-rich N-terminal region, a TIR domain, and a C-terminal region. The TIR domain of TICAM-1 is essential for binding to the TIR domain of TLR3 and also to the TLR4 adaptor TICAM-2 (also called TRIF-related adaptor molecule (TRAM)) (9, 10). Once TICAM-1 is oligomerized, the serine-threonine kinases TANK-binding kinase 1 (TBK1; also called NAK or T2K) and I{kappa}B kinase-related kinase {epsilon} (IKK-{epsilon}; also called IKK-i), are activated and phosphorylate IRF-3 (11, 12). NF-{kappa}B-activating kinase-associated protein 1 (NAP1) participates in the recruitment of these kinases to the N-terminal region of TICAM-1 (13). Whereas the N-terminal region is crucial for TICAM-1-mediated IRF-3 activation, the C-terminal region of TICAM-1 is involved in NF-{kappa}B activation and apoptosis. It has been accepted that receptor-interacting protein 1 (RIP1), a kinase containing a death domain, associates with TICAM-1 via the RIP homotypic interaction motif (RHIM) domain in the C-terminal region and acts as an NF-{kappa}B inducer and apoptosis mediator in TICAM-1-mediated signaling (14, 15, 16).

In contrast to other TLR adaptors, overexpression of TICAM-1 leads to the induction of IFN-β promoter activation and apoptosis. We therefore surmised that the TICAM-1 signaling is initiated apart from TLR3. In this study, we examined the subcellular localization of TICAM-1 in comparison with TLR3 and other signaling molecules in human epithelial cell lines by live cell imaging and confocal immunofluorescence analyses. TICAM-1 appears to be mobile depending on the activation state of its coupling receptor, TLR3. Here we demonstrated that TICAM-1 alters the distribution profile concomitant with its activation from a diffuse cytoplasmic form to a speckle-like structure containing RIP1 and NAP1 where TICAM-1-mediated signaling is initiated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents and cell culture

Anti-human TLR3 mAb (clone TLR3.7) was generated in our laboratory (3). Anti-RIP1 and anti-IRF-3 mAbs were purchased from BD Biosciences. Anti-early endosome Ag 1 (EEA1) polyclonal Ab (pAb) was from Affinity Bioreagents, anti-calnexin pAb was from StressGen Biotechnologies, anti-FLAG M2 mAb, anti-HA pAb, 4',6'-diamidino-2-phenylindole (DAPI) dihydrochloride and benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk) were from Sigma-Aldrich, anti-mannose-6-phosphate receptor (MPR) pAb was from Abcam, anti-Myc mAb was from NeoMarkers (Lab Vision), and anti-p115 pAb was from Calbiochem. LysoTracker Red DND-99 and Alexa Fluor 488- and Alexa Fluor 568-conjugated secondary Abs were from Invitrogen Life Technologies. Polyriboinosinic/polyribocytidylic acid (poly(I:C)) was from Amersham Biosciences, and peptidoglycan (PGN) from Staphylococcus aureus was from Fluka Chemie. Rabbit anti-human TICAM-1 pAb was generated in our laboratory using recombinant TICAM-1 (aa 1–361) as the immunogen. HeLa cells were maintained in MEM (Nissui Pharmaceutical) supplemented with 10% heat-inactivated FCS (Invitrogen Life Technologies).

Plasmids

Complementary DNAs for human TLR3, TICAM-1, and RIP1 were cloned in our laboratory by RT-PCR and ligated at the cloning site of the expression vector, pFLAG-CMV, pEF-BOS, p3xFLAG14 (C-terminal 3xFLAG tag), pECFP-N1 (C-terminal cyan fluorescent protein (CFP) tag), or pEYFP-N1 (C-terminal yellow fluorescent protein (YFP) tag). pECFP-C1 (N-terminal CFP tag) was used to make TICAM-1 tagged with CFP. cDNAs of NAP1 were kindly provided by Dr. M. Nakanishi (Nagoya City University, Nagoya, Japan).

Confocal microscopy

HeLa cells (1.0 x 105 cells/well) were plated onto a micro cover glass (Matsunami Glass) in a 24-well plate. The following day, cells were transfected with the indicated plasmids using Lipofectamine Plus (Invitrogen Life Technologies) or FuGENE HD (Roche). The total amounts of DNA were kept constant by adding empty vector. In some cases transfection was performed in the presence of z-VAD-fmk (final concentration, 20 µM) to inhibit apoptosis. After 24 h (under the presence of z-VAD-fmk) or 12 h (under the absence of z-VAD-fmk), cells were fixed in acetone or 1% formaldehyde in PBS. In the case of formaldehyde fixation, cells were then permeabilized with PBS containing 0.2% Triton X-100 for 15 min. Fixed cells were blocked in PBS containing 1% BSA and labeled with the indicated primary Abs for 1 h at room temperature. Alexa Fluor 488- or Alexa Fluor 568-conjugated secondary Abs (x400 dilution) were used for the visualizing staining of the primary Abs. For acidic organelle staining, cells were treated with LysoTracker (500 µM) for 30 min before fixation. For nucleus staining, cells were treated with DAPI (final concentration, 2 µg/ml) in PBS for 10 min before mounting. After all staining procedures were finished, micro cover glasses were mounted onto a slide glass using PBS containing 2.3% 1,4-diazabicyclo[2.2.2]octane (DABCO) and 50% glycerol. Cells were visualized at x63 magnification under an LSM510 META microscope (Zeiss).

Time lapse analysis

Time-lapse analyses were performed using AquaCosmos 2.0 software (Hamamatsu Photonics) to control a digital imaging system coupled to an inverted microscope (IX-70; Olympus). Briefly, HeLa cells (5 x 105/dish) were plated onto a 36-mm glass dish (IWAKI; Gene Company). The following day, cells were transfected with indicated plasmid using FuGENE HD (Roche) in the presence of z-VAD-fmk. The total amount of DNA (2 µg) was kept constant by adding empty vector. After 18–24 h from transfection, cells were stimulated with 20 µg/ml poly(I:C) or PGN for the indicated times and images were acquired through a cooled charge-coupled device camera (ORCA ER; Hamamatsu Photonics).

Fluorescence resonance energy transfer (FRET) analysis

The sensitized FRET measurement was performed as described previously (17). Fluorescent images were acquired sequentially through YFP, CFP, and FRET filter channels. Filter sets used were YFP (excitation, 500/25 nm; emission, 535/26 nm), CFP (excitation, 440/21 nm; emission, 480/30 nm), and FRET (excitation, 440/21 nm; emission, 535/26 nm). An XF2034 (455DRLP) dichroic mirror was used throughout the experiments. Integration times were 100–200 ms with a 2 x 2 binning mode. The background was subtracted from raw images before FRET calculations. Corrected FRET (FRETC) was calculated on a pixel-by-pixel basis for the entire image using the following equation: FRETC = FRET – (a x CFP) – (b x YFP), where FRET, YFP, and CFP correspond to background-subtracted images of cells coexpressing CFP and YFP acquired through the FRET, YFP, and CFP channels, respectively. a and b are the fractions of bleed-through of CFP and YFP fluorescences, respectively, through the FRET channel that were ordinarily 0.52 and 0.48, respectively, under our experimental conditions.

Reporter gene assay

HeLa cells in a 96-well plate were transiently transfected with the expression vector for TICAM-1YFP (0.1 ng, 10 ng) or empty vector together with p-125 Luc reporter plasmid (15 ng) and an internal control vector, phRL-TK (0.5 ng) using FuGENE HD. The p-125 Luc reporter that contains the human IFN-β promoter region (–125 to + 19) was provided by Dr. T. Taniguchi (University of Tokyo, Tokyo, Japan). The total amount of DNA (100 ng) was kept constant by adding empty vector. After 24 h, cells were lysed in lysis buffer (Promega) and firefly and Renilla luciferase activities were determined according to the manufacturer’s instructions. Luciferase activity was normalized by Renilla luciferase activity and expressed as the fold stimulation relative to the activity in vector-transfected cells.

Immunoblotting

HeLa cells (1 x 105 cells/well) in a 24-well plate were transfected with TICAM-1YFP plasmid (0.5, 50 ng) or empty vector using FuGENE HD in the presence of z-VAD-fmk. The total amount of DNA (500 ng) was kept constant by adding empty vector. After 24 h, cells were lysed in lysis buffer (20 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 1% Nonidet P-40, 10 mM EDTA, 25 mM iodoacetamide, 2 mM PMSF, and protease inhibitor mixture (Roche)) and subjected to SDS-PAGE (10%) under reducing conditions, followed by immunoblotting with rabbit anti-TICAM-1 pAb.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
TLR3 localizes to the early endosome in the human epithelial cell line HeLa

We previously showed that TLR3 localizes to intracellular compartments in human monocyte-derived DCs (7). To identify the organelle in which TLR3 resides, we extended the analysis of the subcellular localization of endogenous TLR3 in HeLa cells with a low level of expression of TLR3 on the plasma membrane and a higher level of expression in intracellular organelles. TLR3-positive compartments were partially merged with EEA1 (Fig. 1). Late endosome (MPR), lysosome (LysoTracker), Golgi (p115), and endoplasmic reticulum (calnexin) markers were not colocalized with TLR3. Thus, in steady-state conditions some TLR3 molecules localize to the early endosome in epithelial cells, consistent with a previous study (18).


Figure 1
View larger version (98K):
[in this window]
[in a new window]

  		FIGURE 1. Subcellular localization of TLR3 in epithelial cell lines. Endogenous TLR3 partly localizes to the early endosome in HeLa cells. Cells were stained with TLR3.7 (20 µg/ml) followed by an Alexa Fluor 488-conjugated secondary Ab. Organelles were stained with anti-EEA1 pAb (2 µg/ml), anti-MPR pAb (10 µg/ml), anti-p115 pAb (3 µg/ml), or anti-calnexin pAb (x200 dilution) followed by an Alexa Fluor 568-conjugated secondary Ab. For LysoTracker staining, cells were pretreated with 500 nM LysoTracker Red for 30 min before fixation (red). Confocal images are shown. The inset in the lower left corner of the EEA-1 merge panel shows a x2-magnified image of the smaller white-boxed area. Green, TLR3; red, organelle markers; blue, nuclei with DAPI; bar, 10 µm.

 
TICAM-1 alters the distribution profile in the cytosol in response to dsRNA

To clarify how TLR3 transmits signals to the adaptor protein TICAM-1, we analyzed the subcellular localization of TICAM-1 in HeLa cells. To visualize the expression of TICAM-1, we prepared a TICAM-1 construct tagged with YFP, TICAM-1YFP, which possesses IRF-3- and NF-{kappa}B- activating abilities. We first coexpressed FLAG-tagged TLR3 and TICAM-1YFP in HeLa cells and subjected these cells to confocal microscopic analysis. As shown in Fig. 2A, overexpressed TICAM-1 was localized to the speckle-like structures in the cytosol and did not colocalize with TLR3 (upper panels). Because overexpressed TICAM-1 induces apoptosis in a caspase-dependent manner, transfected cells were cultured in the presence of a caspase inhibitor (z-VAD-fmk) for 24 h or cultured for only <12 h in the absence of z-VAD-fmk. Under both cell culture conditions TICAM-1 formed a speckle-like localization pattern (data not shown). In contrast, by transfecting a limited dose of the TICAM-1 plasmid into HeLa cells TICAM-1 emerged diffusely in the cytosol and did not colocalize with TLR3 (Fig. 2A, lower panels). In this experimental condition, TICAM-1YFP protein level was so low that it only weekly activated the IFN-β promoter (Fig. 2B, middle column). In contrast, when cells were transfected with a high dose of the TICAM-1YFP plasmid, the TICAM-1YFP protein level was sufficiently high to induce IFN-β promoter activation (Fig. 2B, right column). It has been found that overexpressed TICAM-1 strongly induces IFN-β promoter activation in HEK293 cells irrespective of TLR3 stimulation (5, 19). We therefore hypothesized that the distribution profile of TICAM-1 depends on its activation stage. To test this issue, HeLa cells expressing a low level of TICAM-1YFP were stimulated with poly(I:C), a synthetic dsRNA, and subjected to a time-lapse analysis using an inverted fluorescence microscope. In response to poly(I:C), TICAM-1YFP formed speckle-like structures in a time-dependent manner (Fig. 2C, middle and lower panels). The speckle-like structures appeared 30 min after poly(I:C) stimulation and were gradually increased and concentrated. The tendency of TICAM-1 to form speckles increased to a level comparable to that of overexpression. In the absence of poly(I:C), TICAM-1 remained diffuse in the cytosol (Fig. 2C, upper left panels). Also, TICAM-1 did not alter the distribution profile upon PGN (TLR2 ligand) stimulation, suggesting that TICAM-1 speckle formation specifically depends on TLR3 stimulation (Fig. 2C, upper right panels).


Figure 2
View larger version (70K):
[in this window]
[in a new window]

  		FIGURE 2. The distribution profile of TICAM-1 alters in response to poly(I:C). A, Confocal images of HeLa cells coexpressing TICAM-1YFP and FLAG-TLR3. HeLa cells in a 24-well plate were cotransfected with TICAM-1YFP (50 ng for high expression, 0.5 ng for low expression) and FLAG-TLR3 (250 ng) using FuGENE HD in the presence of Z-VAD-fmk. The total amount of DNA (500 ng) was kept constant by adding empty vector. After 24 h the cells were fixed and TLR3 was stained with anti-FLAG mAb and Alexa Fluor 568-conjugated secondary Ab. Upper panels, HeLa cells were transfected with a high dose of the TICAM-1 plasmid. TICAM-1YFP forms speckle-like structures in the cytosol. Lower panels, HeLa cells were transfected with a low dose of the TICAM-1 plasmid. TICAM-1YFP localizes diffusely in the cytosol. Green, TICAM-1YFP; red, FLAG-TLR3; blue, nuclei with DAPI; bar, 10 µm. B, Upper panel, Luciferase reporter activity of TICAM-1YFP. HeLa cells in a 24-well plate were transiently transfected with TICAM-1YFP plasmid (0.5 ng or 50 ng/well) or empty vector together with the p-125-Luc reporter plasmid and the phRL-TK reporter plasmid using FuGENE HD. After 24 h, luciferase activities were measured and expressed as the fold stimulation relative to the activity in vector-transfected cells. Representative data from a minimum of three separate experiments are shown. Lower panel, Protein expression of TICAM-1YFP was assessed by immunoblotting with anti-TICAM-1 pAb. C, TICAM-1 forms speckle-like structures in response to poly(I:C). HeLa cells expressing a low level of TICAM-1YFP were placed on a time-lapse microscope and stimulated with poly(I:C) (20 µg/ml) (center and lower panels) or PGN (20 µg/ml) (upper right panels) for up to 60 min or left unstimulated (upper left panels). Cell images were taken every 10 min. YFP images at the indicated periods are shown.

 
Transient colocalization of TICAM-1 and TLR3 in response to dsRNA

When poly(I:C) is exogenously added to the cells it is delivered to the intracellular compartments in which TLR3 resides and activates the latter (20, 21). To visualize the association of TICAM-1 with TLR3 upon poly(I:C) stimulation, HeLa cells were cotransfected with TICAM-1YFP and FLAG-TLR3 and stimulated with poly(I:C) for 60 min. Cells were subjected to confocal immunofluorescence analyses. TICAM-1 and TLR3 were localized distinctly in unstimulated cells (Fig. 3). Partial colocalization of TICAM-1 and TLR3 was observed within 30 min after poly(I:C) stimulation. However, 60 min after poly(I:C) stimulation TICAM-1 dissociated from TLR3, forming speckles. Hence, TICAM-1 in the cytosol is transiently recruited to the endosomal TLR3 in response to dsRNA and thereafter moves away from TLR3 to form speckle-like structures.


Figure 3
View larger version (58K):
[in this window]
[in a new window]

  		FIGURE 3. Transient association of TICAM-1 with TLR3 in response to poly(I:C). Confocal images of HeLa cells coexpressing TICAMYFP with FLAG-TLR3 are shown. HeLa cells were cotransfected with TICAMYFP and FLAG-TLR3 and stimulated with poly(I:C) (20 µg/ml) for up to 60 min. At the indicated periods, cells were fixed and stained with anti-FLAG mAb (5 µg/ml) and Alexa Fluor 568-conjugated secondary Ab. Upper panels show x1.5 magnified images of the insets in the lower panels. Green, TICAM-1YFP; red, FLAG-TLR3; blue, nuclei with DAPI; bar, 10 µm.

 
RIP1 and NAP1 are recruited to TICAM-1-positive speckles

To study the relationship between the translocation of TICAM-1 and its functionality, we next analyzed the subcellular localization of the signaling molecules downstream of TICAM-1. RIP1 is crucial for TICAM-1-mediated NF-{kappa}B activation. When RIP1 tagged with YFP (RIP1YFP) was coexpressed with a low dose of TICAM-1 tagged with cyan fluorescent protein (CFP; TICAM-1CFP) in HeLa cells, RIP1YFP was uniformly distributed in the cytosol except for constitutive basal RIP1 accumulation (Fig. 4A, upper panels). Upon poly(I:C) stimulation, RIP1YFP accumulated in the speckle-like structures in a time-dependent manner (Fig. 4A, lower panels). The speckle formation was detected 30 min after poly(I:C) stimulation and then gradually increased up to 60 min, concomitant with TICAM-1 translocation. In the absence of poly(I:C) stimulation, RIP1YFP remained diffusely in the cytosol (Fig. 4A, upper panels).


Figure 4
View larger version (47K):
[in this window]
[in a new window]

  		FIGURE 4. Subcellular localization of RIP1, NAP1, and IRF-3. A and B, RIP1 and NAP1 accumulate in the speckle-like structures in response to poly(I:C). HeLa cells coexpressing RIP1YFP (A) or NAP1YFP (B) with TICAM-1CFP were placed on a time-lapse microscope and imaged every 10 min. Cells were stimulated with poly(I:C) (20 µg/ml) for up to 60 min (lower panels) or left unstimulated (upper panels). Fluorescent images of YFP at the indicated time periods are shown. CFP images indicate the expression level of TICAM-1. C, Nuclear translocation of IRF-3 induced by poly(I:C) stimulation. HeLa cells were seeded onto cover glass and stimulated with poly(I:C) (40 µg/ml) for up to 120 min. At the indicated periods, cells were fixed, stained with anti-IRF-3 mAb and Alexa Fluor 568-conjugated secondary Ab, and subjected to confocal analyses. Green, IRF-3; blue, nuclei with DAPI; bar, 10 µm.

 
Similar results were obtained with NAP1 tagged with YFP (NAP1YFP). NAP1 is located downstream of TICAM-1 and engages in TICAM-1-mediated IRF-3 activation (13). The N-terminal region of TICAM-1 is responsible for the NAP1–TICAM-1 association. When HeLa cells were transfected with NAP1YFP together with TICAM-1CFP and stimulated with poly(I:C), NAP1 formed the speckle-like structures 30 min after poly(I:C) stimulation, as observed in RIP1YFP localization analyses (Fig. 4B). The nuclear translocation of IRF-3 was detected ~60 min after poly(I:C) stimulation, which followed the formation of TICAM-1-positive speckles (Fig. 4C).

These results suggest that TICAM-1, once activated by dsRNA-TLR3, interacts with downstream signaling molecules and forms speckle-like structures in the cytosol apart from TLR3. To carry out a precise examination of the molecular interaction between TICAM-1 and RIP1 or NAP1, these molecules tagged with YFP or CFP were coexpressed in HeLa cells and subjected to FRET analysis after poly(I:C) stimulation. As shown in Fig. 5A, FRETC images revealed a strong energy transfer from RIP1CFP to TICAM-1YFP, whereas energy transfer from NAP1CFP to TICAM-1YFP was relatively weak. These results indicates that RIP1 makes direct contact with TICAM-1 in response to poly(I:C). NAP1 also associates with TICAM-1 in response to poly(I:C), although direct binding was not detected by the yeast two-hybrid technique (data not shown). Thus, TICAM-1 seems to recruit both RIP1 and NAP1 to the putative speckle signalosome. Notably, when TICAM-1 was overexpressed, endogenous RIP1 was highly concentrated in the TICAM-1-positive speckles (Fig. 5B). Also, colocalization of TICAM-1 and NAP1 was partly detected in an overexpression study (Fig. 5C). At a high TICAM-1 expression level, TICAM-1 might be oligomerized in a TLR3-independent manner and activate downstream signaling molecules to form speckle-like structures. These TICAM-1-positive speckles containing RIP1 and NAP1 barely merged with TLR3 or any organelle markers (data not shown). Because recent reports demonstrated that the internalized dsRNA was trafficked with TLR3 from the endosome to the lysosome in both DCs and epithelial cells (20, 21), the TICAM-1 signaling complex appears to reside in the cytosol distinctly from the dsRNA-TLR3-positive compartments.


Figure 5
View larger version (57K):
[in this window]
[in a new window]

  		FIGURE 5. RIP1 and NAP1 are recruited to TICAM-1-positive speckles. A, Interaction of TICAM-1 with RIP1 or NAP1. HeLa cells coexpressing RIP1CFP or NAP1CFP and TICAM-1YFP were stimulated with poly(I:C) (20 µg/ml) for 60 min. FRET, YFP, and CFP images were collected by using an inverted fluorescence microscope equipped with a cooled charge-coupled device camera and FRETc was determined as described in the text. Upper panels show FRET images of RIP1CFP and TICAM-1YFP. The lower graph shows the normalized FRET values of 60 individual regions of cells calculated by dividing FRETC for each region by the mean intensity of RIP1CFP or NAP1CFP. B and C, Confocal images of HeLa cells expressing TICAM-1YFP only (B) or TICAM-1YFP and NAP1-Myc (C). Cells were fixed and stained with anti-RIP1 mAb for detecting endogenous RIP1 (B) or with anti-Myc mAb (C) followed by an Alexa Fluor 568-conjugated secondary Ab. Arrowheads indicate merged speckles. Green, TICAM-1; red, RIP1 or NAP1; blue, nuclei with DAPI; bar, 10 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
TLR signaling is mediated by the recruitment of distinct combinations of adaptor molecules to the cytoplasmic TIR domain of each TLR. Distribution and localization of the adaptor molecules are important factors controlling TLR-mediated signaling. TIRAP/Mal was found to be associated with the plasma membrane via a phosphatidylinositol 4,5-biphosphate-binding domain and facilitates MyD88 delivery to activated TLR4 (22). Also, myristoylation of TICAM-2/TRAM targets it to the plasma membrane and the Golgi apparatus, where it colocalizes with TLR4 (23). Thus, the role of these adaptor proteins other than TICAM-1 has been clearly demonstrated; TIRAP/Mal and TICAM-2/TRAM mainly function as bridges between TLR4 and the signaling adaptors, whereas MyD88 restricts its localization by TIRAP to assemble signal molecules around the TLR4 complex (9, 10, 24, 25). In this study we demonstrated that TICAM-1/TRIF delivers a signal in a unique fashion distinct from that of MyD88.

This is the first study to visualize the molecular dynamics of the TICAM-1 pathway that leads to activation of IRF-3 and NF-{kappa}B. When TICAM-1 expression is controlled to a low level, TICAM-1 emerges diffusely in the cytosol, whereas overexpressed TICAM-1, which strongly induces IFN-β promoter activation and apoptosis, forms speckles. Neither the diffuse nor speckle form of TICAM-1 colocalizes with endogenous and overexpressed TLR3. Once TLR3 is stimulated with dsRNA in cells with diffuse TICAM-1, TICAM-1 is recruited to the TLR3 inside the cells. Thus, TICAM-1 exists unrelated to TLR3 in resting cells. Whereas dsRNA comes over the cells, TICAM-1 is activated along endosomal TLR3 and then the activated TICAM-1 dissociates from the TLR3 to form a speckle-like structure. The speckle-like structure gathers multiple TICAM-1 molecules according to the confocal feature, suggesting the event of the receptor-induced TICAM-1 oligomerization. This TICAM-1 trip facilitates the focusing of the signaling molecules on the speckle containing TICAM-1 and finally activates NF-{kappa}B and IRF-3. In fact, the speckles are comprised of TICAM-1 and the signaling molecules, NAP1 and RIP1. Thus far, however, the intriguing issue as to whether RIP1 and/or NAP1 are recruited to TICAM-1 before or after the dissociation of TICAM-1 from TLR3 remains undetermined. Furthermore, the mechanism by which TICAM-1 is activated by TLR3 also remains undetermined.

TICAM-1 behaves differently from MyD88, which translocates from the cytosol to the membrane and colocalizes with TLR4 or TLR9 upon ligand stimulation (26). In TLR9-mediated signaling in plasmacytoid DCs, MyD88 forms a stable signaling complex containing IL-1 receptor-associated kinase 4 (IRAK4), TNF receptor-associated factor 6 (TRAF6), and IRF-7 on the cytosolic side of TLR9-resided endosomes (27). MyD88 signaling seems to originate at the TLR-containing complex. By contrast, TICAM-1-mediated signaling events are closely associated with the spatiotemporal mobilization and speckle formation of TICAM-1.

TLR3-TICAM-1 signaling results in various cellular responses, including the production of type I IFN and inflammatory cytokines, DC maturation (28), cross-presentation of exogenous Ags for the proliferation of CD8+ T cells (29, 30), NK cell activation (31, 32), and apoptosis (33). TICAM-1 may act as a platform recruiting signaling molecules for DC output. The signaling components participating in the TICAM-1 mediated type I IFN production have been well characterized. Our current focus is to clarify how these components are orchestrated in undefined molecular cascades. It is becoming clear that the TICAM-1 pathway in myeloid DCs is involved in CTL induction and NK cell activation (29, 32). Through these investigations, the cascades by which the TLR3-TICAM-1 pathway induces cross-presentation and NK activation in myeloid DCs will be defined.


   Acknowledgments
 
We thank Drs. M. Shingai, T. Ebihara, A. Ishii, and A. Matsuo in our laboratory for their critical discussions. We also thank Dr. M. Nakanishi (Nagoya City University, Nagoya, Japan) and Dr. T. Taniguchi (Tokyo University, Tokyo, Japan) for providing plasmids.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by Core Research for Engineering, Science, and Technology (CREST), JST (Japan Science and Technology Corporation (JST), grants-in-aid from the Ministry of Education, Science, Sports, and Culture (Specified Project for Advanced Research) and the Hepatitis C Virus (HCV) Project of the National Institute of Health (NIH) of Japan, and by the Uehara Memorial Foundation, Mitsubishi Foundation, Takeda Foundation, and Northtec Foundation. Back

2 Current address: Center for Integrated Medical Research, Keio University, Tokyo, Japan. Back

3 Current address: Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06510. Back

4 Address correspondence and reprint requests to Dr. Misako Matsumoto, Department of Microbiology and Immunology, Hokkaido University Graduate School of Medicine, Kita 15, Nishi 7, Kita-ku Sapporo, Japan. E-mail address: matumoto{at}pop.med.hokudai.ac.jp Back

5 Abbreviations used in this paper: TIR, Toll/IL-1 receptor; CFP, cyan fluorescent protein; DAPI, 4',6'-diamidino-2-phenylindole; DC, dendritic cell; EEA1, early endosome Ag 1; FRET, fluorescence resonance energy transfer; FRETC, corrected FRET; IRF-3, IFN regulatory factor 3; MPR, mannose-6-phosphate receptor; NAP1, NF-{kappa}B activating kinase-associated protein 1; pAb, polyclonal Ab; poly(I:C), polyinosinic/polycytidylic acid; PGN, peptidoglycan; RIP1, receptor-interacting protein 1; TICAM-1, TIR domain-containing adaptor molecule 1; TRAM, TRIF-related adaptor molecule; TRIF, TIR domain-containing adaptor inducing IFN-β; YFP, yellow fluorescent protein; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. Back

Received for publication July 13, 2007. Accepted for publication September 11, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Akira, S., S. Uematsu, O. Takeuchi. 2006. Pathogen recognition and innate immunity. Cell 124: 783-801. [Medline]
  2. Alexopoulou, L., A. C. Holt, R. Medzhitov, R. A. Flavell. 2001. Recognition of double-stranded RNA and activation of NF-{kappa}B by Toll-like receptor 3. Nature 413: 732-738. [Medline]
  3. Matsumoto, M., S. Kikkawa, M. Kohase, K. Miyake, T. Seya. 2002. Establishment of a monoclonal antibody against human Toll-like receptor 3 that blocks double-stranded RNA-mediated signaling. Biochem. Biophys. Res. Commun. 293: 1364-1369. [Medline]
  4. Yoneyama, M., M. Kikuchi, T. Natsukawa, N. Shinobu, T. Imaizumi, M. Miyagishi, K. Taira, S. Akira, T. Fujita. 2004. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat. Immunol. 5: 730-737. [Medline]
  5. Oshiumi, H., M. Matsumoto, K. Funami, T. Akazawa, T. Seya. 2003. TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon-β induction. Nat. Immunol. 4: 161-167. [Medline]
  6. Yamamoto, M., S. Sato, H. Hemmi, K. Hoshino, T. Kaisho, H. Sanjo, O. Takeuchi, M. Sugiyama, M. Okabe, K. Takeda, S. Akira. 2003. Role of adaptor TRIF in the MyD88-independent Toll-like receptor signaling pathway. Science 301: 640-643. [Abstract/Free Full Text]
  7. Matsumoto, M., K. Funami, M. Tanabe, H. Oshiumi, M. Shingai, Y. Seto, A. Yamamoto, T. Seya. 2003. Subcellular localization of Toll-like receptor 3 in human dendritic cells. J. Immunol. 171: 3154-3162. [Abstract/Free Full Text]
  8. Funami, K., M. Matsumoto, H. Oshiumi, T. Akazawa, A. Yamamoto, T. Seya. 2004. The cytoplasmic "linker region" in Toll-like receptor 3 controls receptor localization and signaling. Int. Immunol. 16: 1143-1154. [Abstract/Free Full Text]
  9. Oshiumi, H., M. Sasai, K. Shida, T. Fujita, M. Matsumoto, T. Seya. 2003. TIR-containing adapter molecule (TICAM)-2, a bridging adapter recruiting to Toll-like receptor 4 TICAM-1 that induces interferon-β. J. Biol. Chem. 278: 49751-49762. [Abstract/Free Full Text]
  10. Fitzgerald, K. A., D. C. Rowe, B. J. Barnes, D. R. Caffrey, A. Visintin, E. Latz, B. Monks, P. M. Pitha, D. T. Golenbock. 2003. LPS-TLR4 signaling to IRF-3/7 and NF-{kappa}B involves the Toll adapters TRAM and TRIF. J. Exp. Med. 198: 1043-1055. [Abstract/Free Full Text]
  11. Fitzgerald, K. A., S. M. McWhirter, K. L. Faia, D. C. Rowe, E. Latz, D. T. Golenbock, A. J. Coyle, S. M. Liao, T. Maniatis. 2003. IKK{epsilon} and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 4: 491-496. [Medline]
  12. Sharma, S., B. R. tenOever, N. Grandvaux, G. P. Zhou, R. Lin, J. Hiscott. 2003. Triggering the interferon antiviral response through an IKK-related pathway. Science 300: 1148-1151. [Abstract/Free Full Text]
  13. Sasai, M., H. Oshiumi, M. Matsumoto, N. Inoue, F. Fujita, M. Nakanishi, T. Seya. 2005. Cutting edge: NF-{kappa}B-activating kinase-associated protein 1 participates in TLR3/Toll-IL-1 homology domain-containing adapter molecule-1-mediated IFN regulatory factor 3 activation. J. Immunol. 174: 27-30. [Abstract/Free Full Text]
  14. Meylan, E., K. Burns, K. Hofmann, V. Blancheteau, F. Martinon, M. Kelliher, J. Tschopp. 2004. RIP1 is an essential mediator of Toll-like receptor 3-induced NF-{kappa} B activation. Nat. Immunol. 5: 503-507. [Medline]
  15. Han, K. J., X. Su, L. G. Xu, L. H. Bin, J. Zhang, H. B. Shu. 2004. Mechanisms of the TRIF-induced interferon-stimulated response element and NF-{kappa}B activation and apoptosis pathways. J. Biol. Chem. 279: 15652-15661. [Abstract/Free Full Text]
  16. Kaiser, W. J., M. K. Offermann. 2005. Apoptosis induced by the Toll-like receptor adaptor TRIF is dependent on its receptor interacting protein homotypic interaction motif. J. Immunol. 174: 4942-4952. [Abstract/Free Full Text]
  17. Sorkin, A., M. McClure, F. Huang, R. Carter. 2000. Interaction of EGF receptor and grb2 in living cells visualized by fluorescence resonance energy transfer (FRET) microscopy. Curr. Biol. 10: 1395-1398. [Medline]
  18. Niimi, K., K. Asano, Y. Shiraishi, T. Nakajima, M. Wakaki, J. Kagyo, T. Takihara, Y. Suzuki, K. Fukunaga, T. Shiomi, et al 2007. TLR3-mediated synthesis and release of eotaxin-1/CCL11 from human bronchial smooth muscle cells stimulated with double-stranded RNA. J. Immunol. 178: 489-495. [Abstract/Free Full Text]
  19. Yamamoto, M., S. Sato, K. Mori, K. Hoshino, O. Takeuchi, K. Takeda, S. Akira. 2002. Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-β promoter in the Toll-like receptor signaling. J. Immunol. 169: 6668-6672. [Abstract/Free Full Text]
  20. Lee, H. K., S. Dunzendorfer, K. Soldau, P. S. Tobias. 2006. Double-stranded RNA-mediated TLR3 activation is enhanced by CD14. Immunity 24: 153-163. [Medline]
  21. Johnsen, I. B., T. T. Nguyen, M. Ringdal, A. M. Tryggestad, O. Bakke, E. Lien, T. Espevik, M. W. Anthonsen. 2006. Toll-like receptor 3 associates with c-Src tyrosine kinase on endosomes to initiate antiviral signaling. EMBO J. 25: 3335-3346. [Medline]
  22. Kagan, J. C., R. Medzhitov. 2006. Phosphoinositide-mediated adaptor recruitment controls Toll-like receptor signaling. Cell 125: 943-955. [Medline]
  23. Rowe, D. C., A. F. McGettrick, E. Latz, B. G. Monks, N. J. Gay, M. Yamamoto, S. Akira, L. A. O’Neill, K. A. Fitzgerald, D. T. Golenbock. 2006. The myristoylation of TRIF-related adaptor molecule is essential for Toll-like receptor 4 signal transduction. Proc. Natl. Acad. Sci. USA 103: 6299-6304. [Abstract/Free Full Text]
  24. Yamamoto, M., S. Sato, H. Hemmi, H. Sanjo, S. Uematsu, T. Kaisho, K. Hoshino, O. Takeuchi, M. Kobayashi, T. Fujita, et al 2002. Essential role for TIRAP in activation of the signalling cascade shared by TLR2 and TLR4. Nature 420: 324-329. [Medline]
  25. Horng, T., G. M. Barton, R. A. Flavell, R. Medzhitov. 2002. The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors. Nature 420: 329-333. [Medline]
  26. Ahmad-Nejad, P., H. Hacker, M. Rutz, S. Bauer, R. M. Vabulas, H. Wagner. 2002. Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments. Eur. J. Immunol. 32: 1958-1968. [Medline]
  27. Honda, K., H. Yanai, T. Mizutani, H. Negishi, N. Shimada, N. Suzuki, Y. Ohba, A. Takaoaka, W. C. Yeh, T. Taniguchi. 2005. Role of a transductional-transcriptional processor complex involving MyD88 and IRF-7 in Toll-like receptor signaling. Proc. Natl. Acad. Sci. USA 101: 15416-15421.
  28. Honda, K., S. Sakaguchi, C. Nakajima, A. Watanabe, H. Yanai, M. Matsumoto, T. Ohteki, T. Kaisho, A. Takaoka, S. Akira, et al 2003. Selective contribution of IFN-{alpha}/β signaling to the maturation of dendritic cells induced by double-stranded RNA or viral infection. Proc. Natl. Acad. Sci. USA 100: 10872-10877. [Abstract/Free Full Text]
  29. Schulz, O., S. S. Diebold, M. Chen, T. I. Naslund, M. A. Nolte, L. Alexopoulou, Y. T. Azuma, R. A. Flavell, P. Liljestrom, C. Reis e Sousa. 2005. Toll-like receptor 3 promotes cross-priming to virus-infected cells. Nature 433: 887-892. [Medline]
  30. Salem, M. L., A. N. Kadima, D. J. Cole, W. E. Gillanders. 2005. Defining the antigen-specific T-cell response to vaccination and poly(I:C)/TLR3 signaling: evidence of enhanced primary and memory CD8 T-cell responses and antitumor immunity. J. Immunother. 28: 220-228. [Medline]
  31. Sivori, S., M. Falco, M. Della Chiesa, S. Carlomagno, M. Vitale, L. Moretta, A. Moretta. 2004. CpG and double-stranded RNA trigger human NK cells by Toll-like receptors: induction of cytokine release and cytotoxicity against tumors and dendritic cells. Proc. Natl. Acad. Sci. USA 101: 10116-10121. [Abstract/Free Full Text]
  32. Akazawa, T., T. Ebihara, M. Okuno, Y. Okuda, M. Shingai, K. Tsujimura, T. Takahashi, M. Ikawa, M. Okabe, N. Inoue, et al 2007. Antitumor NK activation induced by the TLR3-TICAM-1 (TRIF) pathway in myeloid dendritic cells. Proc. Natl. Acad. Sci. USA 104: 252-257. [Abstract/Free Full Text]
  33. Salaun, B., I. Coste, M. C. Rissoan, S. J. Lebecque, T. Renno. 2006. TLR3 can directly trigger apoptosis in human cancer cells. J. Immunol. 176: 4894-4901. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
M. Manukyan, P. Nalbant, S. Luxen, K. M. Hahn, and U. G. Knaus
RhoA GTPase Activation by TLR2 and TLR3 Ligands: Connecting via Src to NF-{kappa}B
J. Immunol., March 15, 2009; 182(6): 3522 - 3529.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
K. Itoh, A. Watanabe, K. Funami, T. Seya, and M. Matsumoto
The Clathrin-Mediated Endocytic Pathway Participates in dsRNA-Induced IFN-{beta} Production
J. Immunol., October 15, 2008; 181(8): 5522 - 5529.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Funami, M. Sasai, H. Oshiumi, T. Seya, and M. Matsumoto
Homo-oligomerization Is Essential for Toll/Interleukin-1 Receptor Domain-containing Adaptor Molecule-1-mediated NF-{kappa}B and Interferon Regulatory Factor-3 Activation
J. Biol. Chem., June 27, 2008; 283(26): 18283 - 18291.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Funami, K.
Right arrow Articles by Matsumoto, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Funami, K.
Right arrow Articles by Matsumoto, M.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*Protein
*UniGene
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS