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International Immunology Advance Access originally published online on October 17, 2006
International Immunology 2006 18(12):1701-1706; doi:10.1093/intimm/dxl104
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Fas-associated factor 1 is a negative regulator of PYRIN-containing Apaf-1-like protein 1

Takeshi Kinoshita, Chiaki Kondoh, Mizuho Hasegawa, Ryu Imamura and Takashi Suda

Division of Immunology and Molecular Biology, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa, Ishikawa 920-0934, Japan

Correspondence to: T. Kinoshita; E-mail: tkino{at}kenroku.kanazawa-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
PYRIN-containing apoptotic protease-activating factor 1-like proteins (PYPAFs, also called NALPs) participate in inflammatory signaling by regulating nuclear factor-{kappa}B (NF-{kappa}B) activation and cytokine processing, and have been implicated in autoimmune and inflammatory disorders. However, the precise mechanisms that regulate the signal pathway leading to NF-{kappa}B activation are not completely understood. Here, we used yeast two-hybrid assays to identify Fas-associated factor 1 (FAF1) as a protein interacting with the pyrin domains of several PYPAFs. In these assays, FAF1 interacted strongly with PYPAF1, PYPAF3 and PYPAF7, moderately with PYPAF2 and PYNOD but not at all with the pyrin domains of pyrin or the adaptor molecule apoptosis-associated speck-like protein containing a caspase activation and recruit domain (ASC). The interaction between FAF1 and PYPAF1 in mammalian cells was confirmed by immunoprecipitation assays, and the Fas-interacting domain of FAF1 was critical for this interaction. When co-expressed in HEK293 cells, FAF1 interfere with the NF-{kappa}B activation induced by PYPAF1 and ASC. A FAF1 mutant lacking the Fas-interacting domain showed significantly reduced ability to inhibit NF-{kappa}B activation. In THP-1 cells, the stimulation of NF-{kappa}B up-regulated the level of endogenous FAF1. Taken together, these findings suggest that FAF1 functions as a negative regulator of an NF-{kappa}B signal pathway that involves PYPAF1 and ASC.

Keywords: ASC, inflammation, monocyte, NF-{kappa}B


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Supplementary data
 References
 
PYRIN-containing apoptotic protease-activating factor 1-like proteins (PYPAFs) (also called NALPs) constitute a large subfamily of nucleotide-binding and oligomerization domain-containing proteins that have an N-terminal pyrin-like domain and C-terminal leucine-rich repeats (LRRs) (1–3). Several PYPAFs (such as PYPAF1/cryopyrin, PYPAF5, PYPAF7 and NALP1) induce nuclear factor-{kappa}B (NF-{kappa}B) activation and/or caspase-1-mediated IL-1ß processing in the presence of an adaptor protein, apoptosis-associated speck-like protein containing a caspase activation and recruit domain (ASC) (4–10). Mutations of PYPAF1 are associated with two genetically determined immune disorders, Muckle–Wells syndrome and familial cold auto-inflammatory syndrome (11), indicating that PYPAFs contribute to inflammatory signaling. Compared with the pathway leading to IL-1ß processing, which proceeds through a known series of reactions involving ASC, pro-caspase-1 and pro-IL-1ß, the precise mechanisms that link ASC to NF-{kappa}B activation are not well understood.

Members of the NF-{kappa}B transcription factor family play critical roles in regulating the expression of genes involved in inflammatory and immune responses (12, 13). NF-{kappa}B is activated by various stimuli, including tumor necrosis factor (TNF)-{alpha}, IL-1ß and LPS. In most cells, NF-{kappa}B subunits form a heterocomplex with inhibitor of NF-{kappa}B (I{kappa}B) that sequesters NF-{kappa}B in the cytoplasm in an inactive form. Multiple stimuli activate the I{kappa}B kinase (IKK) complex, which contains catalytic subunits (IKK{alpha} and IKKß) and a regulatory subunit (IKK{gamma}/NEMO); its activation leads to the phosphorylation and degradation of I{kappa}B via the ubiquitin–proteasome pathway. The freed NF-{kappa}B subunits then translocate to the nucleus and activate cellular genes involved in immune responses, inflammation and cell survival (14).

Fas-associated factor 1 (FAF1) was previously identified as a Fas-binding protein (15). When over-expressed, FAF1 induces cell death in some cell types, and it is a component of the death-inducing signaling complex (15–17). FAF1 binds to the death effector domain of FADD and caspase-8 via its death effector domain-interacting domain (DEDID), and binds to the death domain of Fas via its Fas-interacting domain (FID) (16), even though FAF1 does not contain typical death domain motifs. In addition, FAF1 inhibits the NF-{kappa}B activation induced by various stimuli, including TNF-{alpha}, IL-1ß and LPS (18). This inhibition is mediated by the FAF1–DEDID, which physically interacts with the RelA (p65) subunit of NF-{kappa}B and interferes with the nuclear translocation of NF-{kappa}B.

Here, we identified FAF1 as a PYPAF1-interacting protein. When co-expressed, FAF1 interfered with the NF-{kappa}B activation induced by combinational expression of PYPAF1 and ASC in HEK293 cells. The interaction and inhibition were dependent on the FID of FAF1. The level of FAF1 expression in THP-1 was increased in response to LPS stimulation. These findings identify FAF1 as a negative regulator of NF-{kappa}B-activating signal pathway involving PYPAF1 and ASC.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Supplementary data
 References
 
Plasmids
A cDNA encoding FAF1 was amplified by reverse transcription (RT)–PCR from poly A (+) RNA isolated from the THP-1 human monocytic cell line. The cDNAs encoding truncated mutants of FAF1 [FAF1–FID (amino acids 1–180), FAF1–FID + DEDID (amino acids 1–381), FAF1–{Delta}FID (amino acids 181–650) and FAF1–DEDID (amino acids 181–381)] were generated by PCR using FAF1 cDNA as a template and sub-cloned into the pEF-Bos mammalian expression vector (19), in which the FLAG sequence is inserted at the N-terminus of the multi-cloning sites. For plasmid expressing anti-sense oligonucleotides of FAF1, the cDNA encoding amino acids 290–650 were generated by PCR and sub-cloned into pEF-Bos in the reverse orientation. The plasmids expressing human PYPAF1, PYPAF1–{Delta}LRR (amino acids 1–739), pro-caspase-1, pro-IL-1ß, ASC and receptor-interacting protein death domain (RIP-DD, amino acids 558–671) with or without an N-terminal FLAG-tag were described previously (20).

Yeast two-hybrid screening
A LexA-based yeast two-hybrid screen was performed as described previously (21). Briefly, a DNA fragment encoding the PYRIN domain of human PYPAF7 (amino acids 1–88) or PYPAF1 (amino acids 1–90) was cloned into pLexA (Clontech, CA, USA). A cDNA library used for screening was generated from poly (A)+RNA isolated from THP-1 stimulated with LPS or phorbol 12-myristate 13-acetate (PMA)/ionomycin. For the interaction assay, DNA fragments encoding the PYRIN domain of PYPAF3 (amino acids 1–93), PYPAF6 (amino acids 1–86), Pyrin (amino acids 1–93), PYNOD (amino acids 1–100) or ASC (amino acids 1–91) were cloned into pLexA (Clontech) and co-transfected with pB42AD-FAF1.

Cell lines and transfection
The HEK293, HEK293T and THP-1 cell lines were described previously (22). For the luciferase reporter assays and IL-1ß secretion assays, HEK293 cells were plated onto 48-well plates at a density of 4 x 104 cells per well, and transfection was carried out 18 h later using linear polyethyleneimine (molecular weight ~25 kDa, Polysciences Inc., Warrington, PA, USA) (23). For the immunoprecipitation assays, 293T cells were plated onto six-well plates at density of 5 x 105 cells per well, and transfection was carried out 18 h later using linear polyethyleneimine.

Reporter gene assay
NF-{kappa}B assays were described previously (22). HEK293 cells in 48-well plates were transfected with pNF-{kappa}B-Luc firefly reporter (STRATAGENE, La Jolla, CA, USA), pRL-TK renilla reporter (Promega, Madison, WI, USA) and the indicated expression plasmids. The total amount of DNA in each transfection was kept constant (300 ng per well) by the addition of empty vector (pEF-BOS). After 24 h, cell lysates were prepared and the firefly and renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega). The relative luciferase activity (RLA) was calculated as follows: RLA = firefly luciferase activity/renilla luciferase activity.

IL-1ß secretion assay
HEK293 cells were transfected with plasmids encoding pro-IL-1ß, pro-caspase-1 and other expression plasmids for various proteins. The amount of DNA in each transfection was kept constant by the addition of an empty vector. After 26 h, the culture supernatants were collected, and the concentration of IL-1ß was determined using the Human IL-1ß OptEIA ELISA Set (PharMingen, San Diego, CA, USA).

Immunoprecipitation and western blot analysis
Immunoprecipitation and western blotting were carried out as previously described (22), except that mouse anti-FAF1 serum was used for immunoprecipitation and western blotting in some experiments. FAF1-specific antisera were prepared from mice immunized with purified glutathione-S-transferase (GST)-fused FAF1–FID + DEDID (amino acids 1–381).

Assay for mRNA expression
RT–PCR analyses were performed as described previously (22). The primers used to detect human FAF1 mRNA were: sense, 5'-agacagaaatgttgatgtgg-3' and anti-sense, 5'-agcttctaatgagccaataa-3'. The amount of template cDNA was adjusted so that a similar amount of a PCR fragment of ß-actin was generated within the linear range of the PCR.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Supplementary data
 References
 
Isolation of FAF1 as a PYPAF-interacting protein
To gain insight into the molecular mechanisms underlying the pro-inflammatory signal pathway involving the PYPAF family, we screened for PYPAF-interacting proteins using the yeast two-hybrid system. To this end, human cDNA encoding amino acids 1–93 of PYPAF7, which contains the pyrin domain of PYPAF7, was cloned in-frame with the LexA DNA-binding domain and used as ‘bait’ to screen a THP-1 cDNA library. From ~5 million transformants, 11 clones were selected in the primary screening; three of them were found to interact specifically with the pyrin domain of PYAPF7. Upon sequencing, one of these clones was found to contain a partial cDNA of FAF1 (amino acids 29–444). In yeast two-hybrid assays, FAF1 showed a specific interaction with the pyrin domains of PYPAF1, PYPAF3 and PYPAF7 and a weak interaction with PYPAF2 and PYNOD, but it did not interact with the pyrin domains of PYPAF6, PYRIN or ASC (Table 1 and Supplemental Fig. 1, available at International Immunology Online). Of these molecules, PYPAF1 has been implicated in the pathogenesis of human auto-inflammatory diseases. Therefore, we examined the significance of this interaction in the following experiments.


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  		Table 1 Yeast two-hybrid interactions of FAF1 with the pyrin domains of PYPAFs and other pyrin domain-containing proteins

 
FAF1 specifically interacts with PYPAF1 via its FID
The specificity of the interaction between FAF1 with PYPAF1 in mammalian cells was further examined using immunoprecipitation assays (Fig. 1A). FLAG-tagged PYPAF1, a PYPAF1 mutant that lacks the LRR domain (PYPAF1–{Delta}LRR), RIP-DD and XIAP were transiently co-expressed with FAF1 in 293T cells, and cell lysates were prepared for immunoprecipitation assays. FAF1 specifically co-immunoprecipitated with PYPAF1 and PYPAF1–{Delta}LRR, but not with RIP-DD or XIAP. Moreover, the endogenous FAF1 from the 293T cells also co-immunoprecipitated with the exogenous PYPAF1 (Fig. 1B).


Figure 1
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  		Fig. 1 FAF1 interacts with PYPAF1 in vivo. (A) 293T cells in six-well plates were transfected with expression plasmids for FAF1 (0.5 µg) and FLAG–PYPAF1 (1.0 µg), FLAG–PYPAF1–{Delta}LRR (2.0 µg), FLAG–RIP-DD (1.0 µg) or FLAG–XIAP (1.0 µg), as indicated. After 24 h, cell lysates were prepared, and the FLAG-tagged proteins were immunoprecipitated (IP) with anti-FLAG polyclonal antibodies. The immune complexes were analyzed by western blotting (WB) using anti-FAF1 mouse serum (upper panel) or an anti-FLAG mAb (middle panel). A portion of the cell lysates was directly subjected to western blot analysis using anti-FAF1 mouse serum (lower panel). Data are representative of at least three independent experiments. (B) 293T cells in 10-cm dishes were transfected with plasmids encoding FLAG–PYPAF1 (6.0 µg) or FLAG–RIP-DD (1.0 µg), as indicated. After 40 h, cell lysates were prepared and analyzed as above.

 
To investigate which domain of FAF1 is critical for the interaction with PYPAF1, immunoprecipitation assays were performed using a FLAG fusion protein and its truncated mutants (Fig. 2B). FLAG-tagged FAF1, FAF1–{Delta}FID or FID (illustrated in Fig. 2A) was transiently co-expressed with human influenza virus hemagglutinin epitope-tagged PYPAF1 in 293T cells, and cell lysates were prepared for immunoprecipitation assays. PYPAF1 co-immunopreciptated specifically with FAF1 and its FID, but not with FAF–{Delta}FID, indicating that the FID is sufficient for the interaction with PYPAF1. Similar results were obtained in GST pull-down assay using a GST–FAF1 fusion protein and its truncated mutants (data not shown).


Figure 2
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  		Fig. 2 FAF1 interacts with PYPAF1 via its FID. (A) Schematic representations of FAF1 and FAF1 deletion mutants. Locations of FID and DEDID are indicated. Numbers at the ends of each construct represent amino acid numbers. (B) 293T cells in six-well plates were transfected with plasmids encoding human influenza virus hemagglutinin epitope (HA)–PYPAF1 (1.0 µg) and FLAG–FAF1 (1.0 µg), FLAG–FAF1–{Delta}FID (1.0 µg) or FLAG–FAF1–FID (2.0 µg), as indicated. After 24 h, cell lysates were prepared and immunoprecipitated (IP) with the anti-FLAG polyclonal antibody. The immune complexes were analyzed by western blotting (WB) using an anti-HA mAb (upper panel) and an anti-FLAG mAb (middle panel). A portion of the cell lysates was directly subjected to western blot analysis using an anti-HA mAb (lower panel).

 
FAF1 interferes with NF-{kappa}B activation but not the IL-1ß secretion induced by PYPAF1
We first examined whether FAF1 affects the PYPAF1 and ASC-induced NF-{kappa}B activation. As shown in Fig. 3(A), over-expression of FAF1 moderately inhibited TNF-{alpha}-induced NF-{kappa}B activation in 293 cells (by ~30%), and the levels of inhibition by FAF1 were similar to those observed for FAF1–{Delta}FID. This inhibition might be caused by the DEDID, which interferes with the nuclear translocation of RelA downstream of the TNF-{alpha} signaling pathway as reported previously (18). In fact, the levels of inhibition of NF-{kappa}B activation induced by RelA expression were the same for FAF1 and FAF1–{Delta}FID (data not shown). In contrast, FAF1 efficiently inhibited (by ~85%) the NF-{kappa}B activation caused by over-expressing PYPAF1 and ASC in HEK293 cells (Fig. 3B). FAF1–{Delta}FID moderately (by ~45%) inhibited the NF-{kappa}B activation, despite being expressed at the same levels as the exogenous FAF1 and FAF1–{Delta}FID (Fig. 3C). Importantly, the levels of inhibition caused by FAF1–{Delta}FID in this experiment (Fig. 3B) were similar to those it caused in the experiment with TNF-{alpha}-induced NF-{kappa}B activation (Fig. 3A), implying that this inhibition might be also caused by the DEDID.


Figure 3
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  		Fig. 3 PYPAF2 inhibits ASC-mediated NF-{kappa}B activation. (A) HEK293 cells were transfected with the indicated amounts (ng) of expression plasmids encoding FAF1 or FAF1–{Delta}FID together with pNF-{kappa}B-Luc (24 ng) and phRL-TK (12 ng). After 18 h, the cells were or were not treated with TNF-{alpha} (1 ng ml–1) for 6 h. The NF-{kappa}B activity was evaluated by luciferase assays. Error bars represent the range of duplicate samples. Data are representative of at least three independent experiments. (B) HEK293 cells were transfected with the indicated amounts (ng) of expression plasmids encoding FAF1 or FAF1–{Delta}LRR together with PYPAF1–{Delta}LRR (100 ng), ASC (5 ng), pNF-{kappa}B-Luc (24 ng) and phRL-TK (12 ng). After 24 h, the NF-{kappa}B activity was evaluated by luciferase assays. Error bars represent the range of duplicate samples. Data are representative of at least three independent experiments. (C) The cell lysates were subjected to western blot analysis using an anti-FLAG antibody.

 
We next examined whether FAF1 affects the IL-1ß production induced by PYPAF1 and ASC. FAF1 had no effect on the IL-1ß production induced by co-expressing PYPAF1, ASC, pro-caspase-1 and pro-IL-1ß in HEK293 cells (Fig. 4).


Figure 4
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  		Fig. 4 FAF1 does not affect caspase-1-dependent IL-1ß secretion. HEK293 cells were transfected with the indicated amounts (ng) of expression plasmids encoding FAF1 together with the expression plasmids for PYPAF1–{Delta}LRR (25 ng), ASC (5 ng), pro-IL-1ß (150 ng) and pro-caspase-1 (2.5 ng). After 26 h, the culture supernatants were collected and subjected to ELISA for IL-1ß. Error bars represent the range of duplicate samples. Data are representative of at least three independent experiments.

 
Expression of FAF1 is up-regulated in monocytic THP-1 cells by agents that induce NF-{kappa}B activation
Given that FAF1 is a negative regulator of the NF-{kappa}B activation induced by PYPAF1, the expression of FAF1 might be regulated by inflammatory stimuli. To test this hypothesis by a more physiologically relevant system, we examined whether LPS induces the expression of FAF1 in THP-1 cells. As shown in Fig. 5(A), LPS stimulation increased FAF1 mRNA expression, which was detectable as early as 30 min post-stimulation, reached maximal levels by 1 h and declined to basal levels by 2 h. This up-regulation was abrogated by the addition of NF-{kappa}B inhibitor, MG132, confirming that FAF1 up-regulation was actually caused by NF-{kappa}B activation (Supplemental Fig. 2, available at International Immunology Online). A similar induction of FAF1 expression was observed when THP-1 cells were stimulated with PMA (data not shown). Consistently, the expression of FAF1 protein was induced within a few hours after LPS stimulation in THP-1 cells (Fig. 5B).


Figure 5
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  		Fig. 5 Stimulation by LPS induces expression of FAF1 mRNA and protein in THP-1 cells. (A) THP-1 cells were treated with LPS (1 µg ml–1) for indicated times. At each time point, cells were collected and total RNA was prepared. The levels of FAF1 mRNA expression were examined by RT–PCR analysis (upper panel). A ß-actin sequence was amplified as an internal control (lower panel). (B) THP-1 cells were treated with LPS, and cell lysates were prepared. The levels of FAF1 protein were examined by western blot analysis using FAF1-specific antisera.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Supplementary data
 References
 
In this study, we showed that FAF1 binds PYPAF1 and negatively regulates the NF-{kappa}B activation induced by the co-expression of PYAF1 and ASC. FAF1 is reported to inhibit NF-{kappa}B activation induced by various stimuli, including TNF-{alpha}, IL-1ß and LPS (18), and this inhibition is caused by an interaction between the DEDID of FAF1 and RelA that blocks nuclear translocation of RelA. Here, we found that another domain of FAF1, FID, was critical for FAF1's interaction with PYPAF1 and its inhibition of PYPAF1-mediated NF-{kappa}B activation, suggesting that FAF1 regulates NF-{kappa}B activation by a novel mechanism. FAF1 also interacts with PYPAF7, which, like PYPAF1, can induce NF-{kappa}B activation in the presence of ASC (4). Therefore, FAF1 probably inhibits PYPAF7-mediated NF-{kappa}B activation as well.

At present, the precise mechanism by which FAF1 interferes with the NF-{kappa}B signaling pathway downstream of PYPAF1 is not clear. FAF1 expression had no effect on the pro-caspase-1 activation that leads to IL-1ß secretion in HEK293 cells, and the pro-caspase-1 activation is dependent on the assembly of the PYPAF1–ASC complex. Therefore, it seems likely that the interaction of FAF1 with PYPAF1 had no effect on the association of PYPAF1 with ASC. Although the molecular pathway of the ASC-mediated NF-{kappa}B activation is not well elucidated, one possible mechanism is that FAF1's interaction with PYPAF1 inhibits the PYPAF1–NF-{kappa}B signal pathway by interfering with the recruitment or activation of an unidentified factor that links ASC to NF-{kappa}B activation. A candidate for such a FAF1 target is caspase-8, because we recently demonstrated that caspase-8 is involved in the ASC-mediated NF-{kappa}B activation (24), and it was previously reported that FAF1 interacts with caspase-8 via its DEDID (16).

Alternatively, FAF1 may recruit into the PYPAF1–ASC complex, a factor that inhibits the NF-{kappa}B activation pathway, and thereby blocks the activation. Candidates for this inhibitory factor include PYPAF2 and PYNOD, which belong to an anti-inflammatory PYPAF subgroup that has a potential to inhibit NF-{kappa}B activation (PYPAF2, PYPAF4 and PYNOD) (20, 22, 25, 26). In support of this idea, the pyrin domain of PYPAF2 and PYNOD interacted with FAF1 in the yeast two-hybrid assay. Although PYPAF2 showed weaker binding to FAF1 than PYPAF1 in a yeast two-hybrid assay, it showed significant binding to FAF1 in an immunoprecipitation assay using HEK293T cells (data not shown). In this context, it is interesting to note that PYPAF2 has been reported to associate with the IKK complex and inhibit NF-{kappa}B activation (25). Thus, it is tempting to speculate that a PYPAF1–FAF1–PYPAF2 complex sequesters the IKK complex, thereby affecting the activation of NF-{kappa}B. In addition, inflammatory stimuli, including LPS, which induced FAF1 mRNA expression, also increase the endogenous PYPAF2 protein level in THP-1 cells (25), supporting the notion that PYPAF2 participates in a negative feedback loop to blunt the PYPAF1-mediated NF-{kappa}B activation pathways.

The expression of FAF1 in the human monocytic cell line was rapidly induced in response to LPS and PMA, suggesting that the inhibitory complex of PYPAF1 and FAF1 is formed in response to PYPAF1-induced NF-{kappa}B activation and participates in a negative feedback loop to blunt NF-{kappa}B activation in monocytes. The expression of PYPAF1 in peripheral blood cells is primarily restricted to monocytes (5). Therefore, it is tempting to speculate that the scenario described here may be of importance in controlling PYPAF1-mediated NF-{kappa}B activation in monocytes which play a key role in host defense and inflammation.

FAF1 has been shown to inhibit the NF-{kappa}B activation induced by various stimuli, including TNF-{alpha}, IL-1ß and LPS in an HEK293 cell transfection system (18). To examine the physiological suppressive effect of FAF1, we established human monocytic cell lines that stably express anti-sense oligonucleotide of FAF1 cDNA, and examined IL-8 production, a biological marker for NF-{kappa}B activation, of these cells after LPS stimulation. These cell lines with reduced levels of endogenous FAF1 protein produced higher levels of IL-8 than control cell lines (Supplemental Fig. 3, available at International Immunology Online). Although LPS-induced NF-{kappa}B activation does not necessarily involve the formation of FAF1–PYPAF1 complex, these data show the physiological roles of FAF1 in monoytic cells.

In conclusion, we found that FAF1 regulated the PYPAF1–NF-{kappa}B signal via an interaction between FAF1's FID and PYPAF1. We propose that FAF1 functions as a modulator of the PYPAF1-mediated activation of NF-{kappa}B in monocytes.


   Supplementary data
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
References
 
Supplementary figures 1–3 are available at International Immunology Online.


   Acknowledgements
 
This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas (Cancer) from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government and a grant from Novartis Foundation (Japan) for the Promotion of Science.


   Abbreviations
 
ASC, apoptosis-associated speck-like protein containing a caspase activation and recruit domain
DEDID, death effector domain-interacting domain
FAF1, Fas-associated factor 1
FID, Fas-interacting domain
GST, glutathione-S-transferase
I{kappa}B, inhibitor of nuclear factor-{kappa}B
IKK, I{kappa}B kinase
LRR, leucine-rich repeat
NF-{kappa}B, nuclear factor-{kappa}B
PMA, phorbol 12-myristate 13-acetate
PYPAFs, PYRIN-containing apoptotic protease-activating factor 1-like proteins
RIP-DD, receptor-interacting protein death domain
RLA, relative luciferase activity
RT, reverse transcription
TNF, tumor necrosis factor

   Notes
 
Transmitting editor: T. Watanabe

Received 24 August 2005, accepted 14 September 2006.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 

  1. Inohara N and Nunez G. (2003) NODs: intracellular proteins involved in inflammation and apoptosis. Nat. Rev. Immunol. 3:371.[CrossRef][Web of Science][Medline]
  2. Tschopp J, Martinon F, Burns K. (2003) NALPs: a novel protein family involved in inflammation. Nat. Rev. Mol. Cell Biol. 4:95.[CrossRef][Web of Science][Medline]
  3. Stehlik C and Reed JC. (2004) The PYRIN connection: novel players in innate immunity and inflammation. J. Exp. Med. 200:551.[Abstract/Free Full Text]
  4. Wang L, Manji GA, Grenier JM, et al. (2002) PYPAF7, a novel PYRIN-containing Apaf1-like protein that regulates activation of NF-kappa B and caspase-1-dependent cytokine processing. J. Biol. Chem. 277:29874.[Abstract/Free Full Text]
  5. Manji GA, Wang L, Geddes BJ, et al. (2002) PYPAF1, a PYRIN-containing Apaf1-like protein that assembles with ASC and regulates activation of NF-kappa B. J. Biol. Chem. 277:11570.[Abstract/Free Full Text]
  6. Grenier JM, Wang L, Manji GA, et al. (2002) Functional screening of five PYPAF family members identifies PYPAF5 as a novel regulator of NF-kappaB and caspase-1. FEBS Lett. 530:73.[CrossRef][Web of Science][Medline]
  7. Agostini L, Martinon F, Burns K, McDermott MF, Hawkins PN, Tschopp J. (2004) NALP3 forms an IL-1beta-processing inflammasome with increased activity in Muckle-Wells autoinflammatory disorder. Immunity 20:319.[CrossRef][Web of Science][Medline]
  8. Martinon F, Burns K, Tschopp J. (2002) The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol. Cell 10:417.[CrossRef][Web of Science][Medline]
  9. Dowds TA, Masumoto J, Zhu L, Inohara N, Nunez G. (2004) Cryopyrin-induced interleukin 1beta secretion in monocytic cells: enhanced activity of disease-associated mutants and requirement for ASC. J. Biol. Chem. 279:21924.[Abstract/Free Full Text]
  10. Masumoto J, Dowds TA, Schaner P, et al. (2003) ASC is an activating adaptor for NF-kappa B and caspase-8-dependent apoptosis. Biochem. Biophys. Res. Commun. 303:69.[CrossRef][Web of Science][Medline]
  11. Hoffman HM, Mueller JL, Broide DH, Wanderer AA, Kolodner RD. (2001) Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muckle-Wells syndrome. Nat. Genet. 29:301.[CrossRef][Web of Science][Medline]
  12. Beg AA and Baltimore D. (1996) An essential role for NF-kappaB in preventing TNF-alpha-induced cell death. Science 274:782.[Abstract/Free Full Text]
  13. Pahl HL. (1999) Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene 18:6853.[CrossRef][Web of Science][Medline]
  14. Karin M and Ben-Neriah Y. (2000) Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annu. Rev. Immunol. 18:621.[CrossRef][Web of Science][Medline]
  15. Chu K, Niu X, Williams LT. (1995) A Fas-associated protein factor, FAF1, potentiates Fas-mediated apoptosis. Proc. Natl Acad. Sci. USA 92:11894.[Abstract/Free Full Text]
  16. Ryu SW, Lee SJ, Park MY, Jun JI, Jung YK, Kim E. (2003) Fas-associated factor 1, FAF1, is a member of Fas death-inducing signaling complex. J. Biol. Chem. 278:24003.[Abstract/Free Full Text]
  17. Ryu SW, Chae SK, Lee KJ, Kim E. (1999) Identification and characterization of human Fas associated factor 1, hFAF1. Biochem. Biophys. Res. Commun. 262:388.[CrossRef][Web of Science][Medline]
  18. Park MY, Jang HD, Lee SY, Lee KJ, Kim E. (2004) Fas-associated factor-1 inhibits nuclear factor-kappaB (NF-kappaB) activity by interfering with nuclear translocation of the RelA (p65) subunit of NF-kappaB. J. Biol. Chem. 279:2544.[Abstract/Free Full Text]
  19. Mizushima S and Nagata S. (1990) pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res. 18:5322.[Free Full Text]
  20. Wang Y, Hasegawa M, Imamura R, et al. (2004) PYNOD, a novel Apaf-1/CED4-like protein is an inhibitor of ASC and caspase-1. Int. Immunol. 16:777.[Abstract/Free Full Text]
  21. Uekita T, Gotoh I, Kinoshita T, et al. (2004) Membrane-type 1 matrix metalloproteinase cytoplasmic tail-binding protein-1 is a new member of the Cupin superfamily. A possible multifunctional protein acting as an invasion suppressor down-regulated in tumors. J. Biol. Chem. 279:12734.[Abstract/Free Full Text]
  22. Kinoshita T, Wang Y, Hasegawa M, Imamura R, Suda T. (2005) PYPAF3, a PYRIN-containing Apaf-1-like protein, is a feedback regulator of caspase-1-dependent interleukin-1{beta} secretion. J. Biol. Chem. 280:21720.[Abstract/Free Full Text]
  23. Boussif O, Lezoualc'h F, Zanta MA, et al. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl Acad. Sci. USA 92:7297.[Abstract/Free Full Text]
  24. Hasegawa M, Imamura R, Kinoshita T, et al. (2005) ASC-mediated NF-kappaB activation leading to interleukin-8 production requires caspase-8 and is inhibited by CLARP. J. Biol. Chem. 280:15122.[Abstract/Free Full Text]
  25. Bruey JM, Bruey-Sedano N, Newman R, Chandler S, Stehlik C, Reed JC. (2004) PAN1/NALP2/PYPAF2, an inducible inflammatory mediator that regulates NF-kappaB and caspase-1 activation in macrophages. J. Biol. Chem. 279:51897.[Abstract/Free Full Text]
  26. Fiorentino L, Stehlik C, Oliveira V, Ariza ME, Godzik A, Reed JC. (2002) A novel PAAD-containing protein that modulates NF-kappa B induction by cytokines tumor necrosis factor-alpha and interleukin-1beta. J. Biol. Chem. 277:35333.[Abstract/Free Full Text]

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