By
From the * Department of Immunology and Cell Biology, Mario Negri Institute, I-20157 Milan,
Italy; Istituto di I Clinica Medica, Policlinico Umberto I, I-00161 Rome, Italy; and § Department of
Biotechnology, University of Brescia, 25123 Brescia, Italy
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Abstract |
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The human homologue of Drosophila Toll (hToll) is a recently cloned receptor of the interleukin 1 receptor (IL-1R) superfamily, and has been implicated in the activation of adaptive immunity. Signaling by hToll is shown to occur through sequential recruitment of the adapter
molecule MyD88 and the IL-1R-associated kinase. Tumor necrosis factor receptor-activated
factor 6 (TRAF6) and the nuclear factor B (NF-
B)-inducing kinase (NIK) are both involved in subsequent steps of NF-
B activation. Conversely, a dominant negative version of
TRAF6 failed to block hToll-induced activation of stress-activated protein kinase/c-Jun NH2-terminal kinases, thus suggesting an early divergence of the two pathways.
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Introduction |
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Immune response to infection requires the production of cytokines and costimulatory molecules by antigen-presenting cells. Distinct cell-associated receptors on myelomonocytic cells, such as CD14, allow the recognition of pathogen-associated molecules and trigger natural immune response by inducing the production of inflammatory cytokines that subsequently signal to activate adaptive immunity (1).
The molecular mechanisms that control the initial induction of these signals upon infection have been explored recently. In particular, a novel transmembrane receptor homologous to the Drosophila Toll, human Toll (hToll, also called TLR4), has been cloned recently (2, 3). The Drosophila Toll protein controls the potent antifungal response in Drosophila adults (4). Analogously, hToll has been shown to signal activation of adaptive immunity in humans by inducing the expression of B7.1, IL-6, and IL-8; thus, it represents a key molecule for the switching from natural to acquired immunity. However, the biochemical transduction pathway triggered by hToll was ill-defined (2).
hToll is a type I orphan receptor with an extracellular portion containing leucine-rich repeats, and a cytoplasmic domain significantly similar to the intracellular portion of the IL-1R type I (IL-1RI) and the IL-1R accessory protein (IL-1RAcP) (2, 5, 6); these observations suggest that they may use an analogous molecular framework for signaling.
IL-1 triggers the activation of distinct transcription factors, including NF-B and c-Jun/activator protein 1, that
subsequently drive the transcriptional induction of several
cytokine genes (7). The molecular events occurring from
the IL-1R signaling complex to the induction of NF-
B
activity have been characterized recently; in particular, the
adapter protein MyD88 recruits two distinct putative Ser/
Thr kinases, namely IL-1R-associated kinase (IRAK) and
IRAK-2, to the receptor complex (8). IRAK and
IRAK-2 interact subsequently with the adapter molecule
TNFR-activated factor (TRAF) 6, which bridges them to
the protein kinase NF-
B-inducing kinase (NIK) (8, 11,
12). Finally, NIK activates the I-
B kinase complex (including IKK
and IKK
) that directly phosphorylates I-
B
(13).
In this study, we identified and molecularly ordered the
mediators of the hToll-induced NF-B and stress-activated
protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK)
activation cascade.
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Materials and Methods |
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Expression Vectors and Transfection.
TRAF6-Flag,Northern Blotting Analysis.
Monocytes were separated from fresh blood of healthy donors as described previously (19). Total RNA was isolated and analyzed as described previously (19).Coimmunoprecipitation Analysis.
24-36 h after transfection, cells were lysed in 0.5 ml buffer (1% NP-40, 150 mM NaCl, 50 mM Tris, 1 mM EDTA, and protease inhibitor cocktail). Cell lysates were adjusted to 0.7 M NaCl, and the indicated antibodies were added for 1-4 h at 4°C. Immune complexes were precipitated by the addition of protein G-Sepharose (Sigma Chemical Co., St. Louis, MO). After extensive washing (in lysis buffer with the addition of 0.1% SDS), the Sepharose beads were boiled in sample buffer, and eluted proteins were fractionated by SDS-PAGE. Subsequent immunoblotting was performed as described (8).NF-B Activation Assay.
SAPK/JNK Activation Assay.
Cells were transfected with NF- ![]() |
Results and Discussion |
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The expression and eventual regulation of specific transcripts encoding for hToll were analyzed in distinct cell types that play a critical role in the natural immune response. In particular, human monocytes were separated from healthy donors and treated with the bacterial product LPS for different periods of time. As shown in Fig. 1 A, specific transcripts for hToll were present in these cells and were induced significantly after treatment with LPS. These observations suggest that modulation of a nonclonal receptor, namely hToll, after exposure to infectious agents may play a regulatory role in the natural immune response. Of note, PMN and dendritic cells also transcribed hToll mRNA at different levels (our unpublished observations).
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We next analyzed the hToll signaling pathway at the molecular level. A chimeric version of hToll in which the extra
cellular portion was substituted with the corresponding region of CD4 (CD4/hToll) has been shown previously to induce NF-B activation in Jurkat cells (2). We engineered
distinct expression constructs encoding for Flag epitope-
tagged hToll (hToll-Flag) or for a truncated version of hToll
that lacks most of the cytoplasmic portion (
hToll-Flag). Ectopic expression of hToll-Flag but not
hToll-Flag induced NF-
B activation in human embryonic 293T cells at
levels similar to the CD4/Toll chimeric protein that served
as a positive control (Fig. 1, B and C). From these observations, it is apparent that either CD4-driven or ectopic expression-forced aggregation of the cytoplasmic portions of
distinct hToll receptors is sufficient to trigger a signaling cascade that leads ultimately to activation of the transcription
factor NF-
B. Given this, one could speculate that an as yet
unidentified hToll ligand binds to hToll and induces its oligomerization and subsequent signaling cascade, in a manner similar to IL-1 and TNF with their cognate receptors.
hToll shares significant sequence similarity with distinct
members of the IL-1R family, including IL-1RI, IL-1RAcP,
and MyD88; of note, Phe 513 and Trp 514 in IL-1RI, which
are conserved in all of these proteins, have been shown to
be essential for IL-1RI to signal (Fig. 2 A) (20). Since we
have shown recently a homophilic interaction to occur between the IL-1RAcP and MyD88 throughout their homologous domains (8), we asked whether hToll and the adapter protein MyD88 could interact. Upon coexpression,
MyD88 and hToll formed an immunoprecipitable complex; in contrast, a mutant version of hToll, which lacks the
region of homology to MyD88 and which was unable to
induce NF-B activation, failed to bind MyD88 (Fig. 2 B).
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A mutant version of MyD88 (MyD88), encoding only
for the COOH-terminal IL-1R-like domain, abrogates IL-1RI/IL-1RAcP-induced NF-
B activation (8); given this,
we analyzed whether
MyD88 could act as a dominant
negative inhibitor of hToll-induced NF-
B activation. As
predicted,
MyD88 specifically inhibited hToll-induced but not TNFR-2-induced NF-
B activity, lending functional credence to the interaction occurring between hToll
and MyD88 (Fig. 2 C). From these observations, it is apparent that both IL-1R and hToll recruit the adapter protein MyD88 to their respective signaling complex.
IRAK and IRAK-2 are two additional proximal mediators of the IL-1R signaling complex; IRAK is recruited to the IL-1RAcP, whereas IRAK-2 preferentially binds IL-1RI (8, 10). Given this, we asked whether IRAK or IRAK-2 could interact with hToll. Upon ectopic expression, IRAK and hToll formed an immunoprecipitable complex. In contrast, IRAK-2 bound only weakly to hToll compared with IL-1RI (Fig. 3, A and B), thus suggesting that it may not represent a relevant hToll signal transducer.
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NF-B activation induced by a number of cytokine receptors is mediated by members of the TRAF adapter family. TRAF2, for example, plays a critical role in NF-
B activation mediated by TNFR-1 and TNFR-2 (21, 22).
TRAF6 has been implicated in the IL-1 signaling pathway
and has been shown to complex with IRAK and IRAK-2
downstream to the receptor signaling complex (8, 11).
Therefore, we determined whether dominant negative versions of either could act to inhibit hToll-induced NF-
B
activity.
TRAF6 but not
TRAF2 significantly impaired
hToll-induced NF-
B activity, suggesting that TRAF6
may act as an additional downstream mediator of the hToll-induced NF-
B activation cascade (Fig. 3 C).
The protein kinase NIK has been shown to act as a general mediator of TRAF-induced NF-B activation (12);
once activated, NIK directly phosphorylates and activates
the IKK
/
complex, which is responsible for I-
B
phosphorylation and subsequent NF-
B activation (13).
Dominant negative versions of NIK, in which the critical
lysine has been mutated to alanine [NIK 429-430 (KK-AA)], act to inhibit NF-
B activation induced by Fas,
TNF, and IL-1 (12). Given this, we asked whether
NIK(KK-AA) could inhibit hToll-induced NF-
B activation. NIK(KK-AA) abrogated NF-
B activity triggered by
hToll ectopic expression (Fig. 3 C) as well as by TRAF6
overexpression (not shown).
In addition to inducing activation of NF-B, distinct inflammatory cytokines also induce SAPK, also known as
JNK. The active phosphorylated form of SAPK binds to
and phosphorylates the transcription factors c-Jun, activating transcription factor 2 (ATF2), and ternary complex factor (TCF)/Elk1 (23). In particular, activation of SAPK/
JNK by the TNFR-1 occurs through a TRAF2-dependent
pathway, as a dominant negative version of TRAF2 acts to
inhibit both NF-
B and c-Jun activation induced by TNF
(18, 26, 27). In contrast, a dominant negative version of
NIK, which abrogates TNF-induced NF-
B activation,
fails to inhibit c-Jun phosphorylation, supporting a model
wherein TNF-mediated NF-
B and c-Jun pathways bifurcate at TRAF2 (28, 29). Therefore, we analyzed whether
hToll induced SAPK/JNK. Ectopic expression of increasing amounts of hToll-Flag but not
hToll-Flag resulted in
activation of SAPK
as indicated by specific phosphorylation at Thr 183 and Tyr 185 (Fig. 4 A). Overexpression of
TRAF6 also activated SAPK as described previously (28).
To identify mediators of hToll-mediated JNK activation, we
cotransfected 293 cells with hToll and dominant negative
versions of either MyD88 or TRAF6. Surprisingly,
MyD88 but not
TRA6 acted to inhibit hToll-triggered
JNK phosphorylation (Fig. 4 B). Importantly, under the
same experimental conditions, both
MyD88 and
TRAF6 abrogated hToll-induced NF-
B activation (96 and 90% inhibition, respectively). Additionally,
TRAF6 alone, as well
as
MyD88, failed to activate JNK (data not shown). Collectively, these observations indicate that although ectopic
expression of TRAF6 induced SAPK, a dominant negative
version of TRAF6 failed to inhibit hToll-induced JNK
phosphorylation. Given this, one could speculate that although TRAF6 overexpression is sufficient to activate JNK/
SAPK, endogenous TRAF6 does not provide a significant
contribution to JNK/SAPK activation by hToll (Fig. 5).
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Regardless, MyD88 appears to represent the most upstream mediator of the hToll-mediated signaling cascade,
which ultimately activates NF-B and c-Jun, thus driving
transcriptional activation of several cytokines and costimulatory molecules. Therefore, it may represent a potentially
useful therapeutic target for controlling the molecular
switch from the innate to the adaptive immune response.
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Footnotes |
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Address correspondence to Marta Muzio, Dept. Immunology and Cell Biology, Mario Negri Institute, via Eritrea 62, I-20157, Milano, Italy. Phone: 39-2-39014532; Fax: 39-2-3546277; E-mail: muziom{at}irfmn.mnegri.it
Received for publication 26 February 1998 and in revised form 9 April 1998.
M. Muzio is supported by Federazione Italiana Ricerca sul Cancro. This work was supported by European Community Special Project Biotechnology, Consiglio Nazionale Ricerche, and Associazione Italiana Ricerca sul Cancro.We wish to thank R. Medzhitov and C.A. Janeway for CD4/Toll cDNA, James Woodgett for HA-SAPK-pCDNA3, and Z. Cao for reagents and for critical reading of the manuscript.
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References |
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![]() ![]() ![]() ![]() ![]() ![]() |
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1. | Medzhitov, R., and A. Janeway Jr.. 1997. Innate immunity: the virtues of a nonclonal system of recognition. Cell. 91: 295-298 [Medline]. |
2. | Medzhitov, R., P. Preston-Hurlburt, and C.A. Janeway. 1997. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature. 388: 394-397 [Medline]. |
3. | Rock, F.L., G. Hardiman, J.C. Timans, R. Kastelein, and F.J. Bazan. 1997. A family of human receptors structurally related to Drosophila Toll. Proc. Natl. Acad. Sci. USA. 95: 558-592 . |
4. | Lemaitre, B., E. Nicolas, L. Michaut, J.-M. Reichart, and J.A. Hoffmann. 1996. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell. 86: 973-983 [Medline]. |
5. |
Greenfeder, S.A.,
P. Nunes,
L. Kwee,
M. Labow,
R.A. Chizzonite, and
G. Ju.
1995.
Molecular cloning and characterization of a second subunit of the interleukin 1 receptor
complex.
J. Biol. Chem.
270:
13757-13765
|
6. | Gay, N.J., and F.J. Keith. 1991. Drosophila Toll and IL-1 receptor. Nature. 351: 355-356 [Medline]. |
7. |
Dinarello, C.A..
1996.
Biologic basis for interleukin-1 in disease.
Blood.
87:
2095-2147
|
8. |
Muzio, M.,
J. Ni,
P. Feng, and
V.M. Dixit.
1997.
IRAK
(Pelle) family member IRAK-2 and MyD88 as proximal mediators of IL-1 signaling.
Science.
278:
1612-1615
|
9. | Wesche, H., W.J. Henzel, W. Shillinglaw, S. Li, and Z. Cao. 1997. MyD88: an adapter that recruits IRAK to the IL-1 receptor complex. Immunity. 7: 837-847 [Medline]. |
10. | Cao, Z., W. Henzel, and X. Gao. 1996. IRAK: a kinase associated with the interleukin-1 receptor. Science. 271: 1128-1131 [Abstract]. |
11. | Cao, Z., J. Xiong, M. Takeuchi, T. Kurama, and D. Goeddel. 1996. TRAF6 is a signal transducer for interleukin-1. Nature. 383: 443-446 [Medline]. |
12. |
Malinin, N.L.,
M.P. Boldin,
A.V. Kovalenko, and
D. Wallach.
1997.
MAP3K-related kinase involved in NF-![]() |
13. |
DiDonato, J.A.,
M. Hayakawa,
D.M. Rothwarf,
E. Zandi, and
M. Karin.
1997.
A cytokine-responsive I![]() ![]() |
14. |
Régnier, C.H.,
H.Y. Song,
X. Gao,
D.V. Goeddel,
Z. Cao, and
M. Rothe.
1997.
Identification and characterization of an
I![]() |
15. |
Mercurio, F.,
H. Zhu,
B.W. Murray,
A. Shevcenko,
B.L. Bennet,
J.W. Li,
D.B. Young,
M. Barbosa,
M. Mann,
A. Manning, and
A. Rao.
1997.
IKK-1 and IKK-2: cytokine-activated I![]() ![]() |
16. |
Woronicz, J.D.,
X. Gao,
Z. Cao,
M. Rothe, and
D. Goeddel.
1997.
I![]() ![]() ![]() ![]() ![]() |
17. |
Zandi, E.,
D.M. Rothwarf,
M. Delhase,
M. Hayakawa, and
M. Karin.
1997.
The I![]() ![]() ![]() ![]() ![]() |
18. |
Natoli, G.,
A. Costanzo,
A. Ianni,
D.J. Templeton,
J.R. Woodgett,
C. Balsano, and
M. Levrero.
1997.
Activation of
SAPK/JNK by TNF receptor-1 through a noncytotoxic
TRAF2-dependent pathway.
Science.
275:
200-203
|
19. |
Muzio, M.,
F. Re,
M. Sironi,
N. Polentarutti,
A. Minty,
D. Caput,
P. Ferrara,
A. Mantovani, and
F. Colotta.
1994.
Interleukin-13 induces the production of interleukin-1 receptor
antagonist (IL-1ra) and the expression of the mRNA for the
intracellular (keratinocyte) form of IL-1ra in human myelomonocytic cells.
Blood.
83:
1738-1743
|
20. |
Heguy, A.,
C.T. Baldari,
G. Macchia,
J.L. Telford, and
M. Melli.
1992.
Amino acids conserved in interleukin-1 receptors (IL-1Rs) and the Drosophila Toll protein are essential for
IL-1R signal transduction.
J. Biol. Chem
267:
2605-2609
|
21. | Rothe, M., S.C. Wong, W.J. Henzel, and D.V. Goeddel. 1994. A novel family of putative signal transducers associated with the cytoplasmic domain of the 75kDa tumor necrosis factor receptor. Cell. 78: 681-692 [Medline]. |
22. |
Rothe, M.,
V. Sarma,
V.M. Dixit, and
D.V. Goeddel.
1995.
TRAF2-mediated activation of NF-![]() |
23. | Derijard, B., M. Hibi, I.-H. Wu, T. Baret, B. Su, T. Deng, M. Karin, and R. Davis. 1994. JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell. 76: 1025-1037 [Medline]. |
24. | Kyriakis, J.M., P. Banerjee, E. Nikolakaki, T. Dai, E.A. Rubie, M.F. Ahmad, J. Avruch, and J.R. Woodgett. 1994. The stress-activated protein kinase subfamily of c-Jun kinases. Nature. 369: 156-160 [Medline]. |
25. |
Adler, V.,
A. Polotskaya,
F. Wagner, and
A.S. Kraft.
1992.
Affinity-purified c-Jun amino-terminal protein kinase requires serine/threonine phosphorylation for activity.
J. Biol.
Chem
267:
17001-17005
|
26. |
Reinhard, C.,
B. Shamoon,
V. Shyamala, and
L.T. Williams.
1997.
Tumor necrosis factor ![]() |
27. |
Liu, Z.,
H. Hsu,
D.V. Goeddel, and
M. Karin.
1996.
Dissection of TNF receptor 1 effector functions: JNK activation is
not linked to apoptosis while NF-![]() |
28. |
Song, H.J.,
C.H. Régnier,
C.J. Kirschining,
D.V. Goeddel, and
M. Rothe.
1997.
Tumor necrosis factor (TNF)-mediated
kinase cascades: bifurcation of NF-![]() |
29. |
Natoli, G.,
A. Costanzo,
F. Moretti,
M. Fulco,
C. Balsano, and
M. Levrero.
1997.
TNF receptor-1 signaling downstream of TRAF2.
J. Biol. Chem.
272:
26079-26082
|