Toll-like Receptors 2 and 4 Activate STAT1 Serine Phosphorylation by Distinct Mechanisms in Macrophages*

Sang Hoon Rhee {ddagger}, Bryan W. Jones {ddagger}, Vladimir Toshchakov §, Stefanie N. Vogel § and Matthew J. Fenton {ddagger} 

From the {ddagger}The Pulmonary Center, Boston University School of Medicine, Boston, Massachusetts 02118 and §Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 21201

Received for publication, August 22, 2002 , and in revised form, April 4, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Engagement of Toll-like receptor (TLR) proteins activates multiple signal transduction pathways. These studies show that engagement of TLR2 and TLR4 leads to rapid phosphorylation of the transcription factor STAT1 at serine 727 (Ser-727 STAT1) in murine macrophages. Only TLR4 engagement induced STAT1 phosphorylation at tyrosine 701, although this response was delayed compared with Ser-727 STAT1 phosphorylation. Inhibition of phosphatidylinositol 3'-kinase using LY294002 blocked TLR4-induced STAT1 tyrosine phosphorylation, but this inhibitor had no effect on STAT1 serine phosphorylation. TLR-induced phosphorylation of Ser-727 STAT1 could be blocked by the selective p38 mitogen-activated protein kinase inhibitor SB203580. However, activation of p38 was not sufficient to induce Ser-727 STAT1 phosphorylation in macrophages. TLR2-induced activation of Ser-727 STAT1 phosphorylation required the adapter protein MyD88, whereas TLR4-induced activation of Ser-727 STAT1 phosphorylation was not solely dependent on MyD88. Lastly, TLR4-induced activation of Ser-727 STAT1 phosphorylation could be blocked by rottlerin, a specific inhibitor of protein kinase C-{delta}. In contrast, rottlerin had no effect on STAT1 phosphorylation induced via TLR2. Together, these data demonstrate that activation STAT1 tyrosine and serine phosphorylation are distinct consequences of TLR engagement in murine macrophages. Furthermore, p38 mitogen-activated protein kinase, protein kinase C-{delta}, and a novel TLR2-specific signaling pathway appear to be necessary to induce Ser-727 STAT1 phosphorylation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian Toll-like receptors (TLRs)1 are type I transmembrane receptors that are composed of an extracellular leucine-rich repeat domain and a highly conserved cytoplasmic Toll/IL-1R domain (1). These receptors are expressed on a variety of cell types, including dendritic cells, macrophages, endothelial cells, lymphocytes, and epithelial cells. TLR proteins are primary signal-transducing molecules responsible for recognizing specific microbial pathogen-associated molecular patterns, including Gram-negative bacterial lipopolysaccharide (LPS) (2). A variety of diverse chemical structures has been identified for most of the ten known TLR proteins (reviewed in Ref. 3). The activation of TLR proteins is believed to give rise to patterns of gene expression that are necessary to initiate both innate and adaptive immunity (4, 5). TLR agonists are known to activate multiple signal transduction pathways simultaneously in target cells. These signaling events include activation of the transcription factors NF-{kappa}B and AP-1, the MAP kinases, protein kinase C isoforms, and the lipid kinase phosphatidylinositol 3'-kinase (PI3K) (69).

Another early signaling event in LPS-stimulated macrophages is the activation of the transcription factor STAT1 (signal transducer and activator of transcription 1). Various cytokine receptors exploit STAT proteins to transduce ligand-induced signaling. The type I interferons (IFN-{alpha}/{beta}) utilize STAT1 and STAT2 to transduce intracellular signals generated following engagement of a heterodimeric receptor complex consisting of the subunit chains, IFNAR-1 and IFNAR-2 (10, 11). IFN-{alpha}/{beta} receptor results in the cross-activation of the two receptor-associated Janus protein tyrosine kinases (Jaks), Tyk2 and Jak1, respectively. Thereby, activated Tyks and Jaks lead to the phosphorylation on Tyr-701 in STAT1 and STAT2, resulting in homodimeric (STAT1·STAT1), heterodimeric (STAT1·STAT2), or heterotrimeric (STAT1·STAT2·interferon regulatory factor-9) protein complexes. These multimeric complexes translocate to the nucleus where they bind to distinct DNA elements, finally leading to the activation of IFN-inducible gene expression (10, 12). Phosphorylation of Tyr-701 alone is sufficient to generate STAT multimers that possess DNA binding activity (15), although phosphorylation of Ser-727 is required for maximal transcriptional activity of STAT1 (reviewed in Ref. 14). However, the signaling mechanisms leading to STAT1 Ser-727 phosphorylation are not well understood. Several studies have reported that the p38 MAP kinase is necessary for STAT1 phosphorylation at Ser-727, but it is likely that STAT1 is not a substrate for p38 in living cells (14).

The studies presented below show that LPS induced TLR4-dependent phosphorylation of STAT1 at both tyrosine and serine residues in murine macrophages. LPS-induced STAT1 Tyr-701 phosphorylation was mediated by a PI3K-dependent mechanism, whereas STAT1 Ser-727 phosphorylation was PI3K-independent. In contrast, macrophage activation via TLR2 induced phosphorylation of Ser-727, but not of Tyr-701, on STAT1. Furthermore, the adapter protein MyD88 was found to be necessary for STAT1 serine phosphorylation via TLR2 but not via TLR4. The p38 MAP kinase was necessary but was not sufficient for TLR-dependent activation of STAT1 Ser-727 phosphorylation. Moreover, specific inhibitors of PKC-{delta} were found to block STAT1 Ser-727 phosphorylation induced via TLR4 but not via TLR2. These studies revealed a novel difference in the mechanism of STAT1 phosphorylation induced by engagement of TLR2 and TLR4.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Sources of Macrophages—LPS-hyporesponsive C3H/HeJ mice and normal C3H/OuJ mice were purchased from Jackson Laboratories (Bar Harbor, ME). TLR2/ and MyD88/ mice were provided by Dr. Shuzio Akira (University of Osaka Medical School, Osaka, Japan) and have been described previously (16). These mice were back-crossed into a C57BL/6 background for four generations prior to use. C57BL/6 mice from Jackson Laboratories were used as controls for the TLR2-deficient mice. Primary peritoneal macrophages were prepared from these mice using thioglycollate elicitation as described previously (17). The murine macrophage RAW264.7 cell line (ATCC TIB-71; American Type Culture Collection, Manassas, VA) were cultured in LPS-free Dulbecco's modified Eagle's medium containing 10% (v/v) heat-inactivated fetal bovine serum, 1% L-glutamine, and 10 units/ml penicillin and 100 µg/ml streptomycin (Invitrogen) at 37 °C in air supplemented with 5% CO2.

Plasmids and Reagents—The constitutively active form of murine TLR4 was described previously (6). The dominant-negative p85 expression plasmid was kindly provided by Dr. Julian Downward (Imperial Cancer Research Fund, London, United Kingdom) and was described previously (18). The PKC-{delta} dominant-negative expression plasmid was provided by Dr. Michael Simons (Dartmouth Medical School) and was described previously (19). The human IFN-{beta} promoter luciferase reporter plasmid was provided by Dr. John Hiscott (McGill University, Montreal, Quebec, Canada) and was described previously (20). The murine COX2 promoter luciferase reporter plasmid was provided by Dr. Daniel Hwang (University of California, Davis, CA) and was also described previously (6). All plasmids were prepared using the EndoFree plasmid kit as recommended by the manufacturer (Qiagen, Valencia, CA). Highly purified protein-free Escherichia coli K235 LPS was prepared as described by Hirschfeld et al. (21). The synthetic lipopeptide Pam3Cys (S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-Lys4-OH trihydrochloride) was from EMC Microcollections GmbH (Tubingen, Germany). LY294002 was from Sigma. Bisindoylmaleamide, rottlerin, and SB203580 were purchased from Calbiochem. Recombinant murine IFN-{gamma} was purchased from R & D Systems (Minneapolis, MN), and recombinant murine IL-1{beta} was purchased from Peprotech (Rocky Hill, NJ). Antibodies against Akt, Ser-473-phosphorylated Akt, STAT1, Tyr-701-phosphorylated STAT1, and phosphorylated MAP kinase p38 were from Cell Signaling Technology (Beverly, MA). The antibody recognizing Ser-727-phosphorylated STAT1 was from Upstate Biotechnology (Lake Placid, NY).

Transfection and Luciferase Reporter Assays—RAW264.7 cells were plated in six-well plates (1.2 x 106 cells/well) and transfected with the appropriate plasmid DNA, including a {beta}-galactosidase expression plasmid (HSP70-{beta}-gal) as an internal control, using SuperFect transfect reagent (Qiagen) according to the manufacturer's instruction. One day after transfection, relative luciferase activity was determined by normalization with {beta}-galactosidase activity as described previously (6, 22). All assay were performed in triplicate, and a single representative experiment is shown. Data are expressed as mean values ± S.E.

Western Blot Analysis—Cells were harvested and washed once with phosphate-buffered saline, pH 7.5, and then lysed for 30 min on ice in lysis buffer (150 mM NaCl, 50 mM Tris-Cl, pH 8.0, 5 mM EDTA, 1% Nonidet P-40) with protease inhibitor mixture (Roche Applied Science) and phosphatase inhibitor mixture (Sigma). Cell lysates were clarified by centrifugation at 4 °C for 10 min at 12,000 x g. Protein concentrations of the lysate were measured by Bradford method (Bio-Rad), and an equal amount of total protein per lane was fractionated on a 10% SDS-polyacrylamide gel using Laemmli sample buffer (23). Gels were transferred to polyvinylidene difluoride membranes. The membranes were blocked with Tris-buffered saline containing 0.05% Tween 20 and 5% nonfat dry milk and then incubated with the indicated antibodies and an appropriate horseradish peroxidase-conjugated secondary antibody as described elsewhere (6). Bound antibodies were visualized using the enhanced chemiluminescence system (Pierce).

Reverse Transcriptase PCR—Semi-quantitative reverse transcriptase PCR amplification of total RNA was performed as described previously (20). The oligonucleotide primers used for amplification of the murine IFN-{beta} PCR product were 5'-TCCAAGAAAGGACGAACATTCG-3' and 5'-TGAGGACATCTCCCACGTCAA-3' (annealing temperature, 55 °C).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
TLR Engagement Leads to Phosphorylation of STAT1—Previous reports have demonstrated that TLR signaling leads to the activation of MAP kinases and the transcriptions factors NF-{kappa}B and AP-1 in macrophages (6, 7). Subsequent studies sought to determine whether engagement of TLR proteins also leads to the activation of the transcription factor STAT1. RAW264.7 murine macrophages were stimulated with the TLR4 agonist E. coli LPS, or with the synthetic lipopeptide TLR2 agonist Pam3Cys, for various times. Whole cell lysates were prepared, and lysates were then analyzed by Western blotting. STAT1 activation was measured using specific anti-bodies that discriminate between STAT1 phosphorylated at serine 727 and tyrosine 701. As shown in Fig. 1A, E. coli LPS was capable of inducing the phosphorylation of STAT1 at both serine and tyrosine residues. Pam3Cys was capable of inducing STAT1 Ser-727 phosphorylation with similar kinetics to LPS (Fig. 1B), but this TLR2 agonist was incapable of inducing STAT1 tyrosine phosphorylation (data not shown). The inability of TLR2 agonists to induce STAT1 tyrosine phosphorylation has been reported previously (20) and is because of the inability of TLR2 agonists to induce IFN-{beta} secretion and subsequent engagement of the type I IFN receptor.



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FIG. 1.
Kinetics of TLR-dependent STAT1 phosphorylation in RAW264.7 macrophages. A, RAW264.7 murine macrophages (3 x 106 cells) were plated in 6-well plates 1 day prior to the experiment. Cells were stimulated with E. coli LPS (100 ng/ml) for the indicated times. The cell lysates were fractionated by SDS-PAGE and analyzed by Western blotting using an antibody against phospho-Ser-727 (P-S(727)) and phospho-Tyr-701 (P-Y(701)) STAT1. Membranes were then stripped and re-probed using an antibody against total STAT1 and against {beta}-actin as control for equal loading. In parallel dishes, RAW264.7 cells were stimulated with IFN-{gamma} (200 units/ml, 4 h) as a positive control for phosphorylation of STAT1. B, RAW264.7 macrophages were cultured as described above and then stimulated with the synthetic lipopeptide Pam3Cys (1 µg/ml) for the indicated times. The cell lysates were fractionated by SDS-PAGE and analyzed by Western blotting using an antibody against the phospho-Ser-727 STAT1 or against total STAT1 as indicated.

 

The kinetics of STAT1 serine phosphorylation were rapid and sustained, with maximal serine phosphorylation occurring in less than 30 min, whereas STAT1 tyrosine phosphorylation was both delayed and transient. Two distinct species of tyrosine-phosphorylated STAT1 were observed, corresponding to STAT1{alpha} (92 kDa) and STAT1{beta} (84 kDa). A single species of serine-phosphorylated STAT1 was observed, corresponding to STAT1{alpha}. The STAT1{beta} splice variant lacks the C-terminal serine phosphorylation site present in STAT1{alpha}. In the figures, this serine-phosphorylated form of STAT1{alpha} will simply be referred to as P-S(727)STAT1. STAT1 tyrosine phosphorylation was first observed ~2 h after LPS stimulation, as reported previously (20), and was substantially diminished by 10 h after LPS stimulation. This reduction in STAT1 tyrosine phosphorylation coincided with an overall increase in total STAT1 levels and may reflect an overall increase in the total cellular content of STAT1. Alternatively, transient STAT1 tyrosine phosphorylation may reflect the action of protein tyrosine phosphatases. These data reveal that engagement of TLR2 and TLR4 leads to the rapid serine phosphorylation of STAT1. Moreover, the distinct kinetics of STAT1 serine and tyrosine phosphorylation suggests that these events are consequences of distinct signal transduction pathways.

To determine whether TLR4 was necessary for the activation of STAT1 serine phosphorylation, peritoneal macrophages from normal C3H/OuJ and TLR4 mutant C3H/HeJ mice were stimulated in vitro with E. coli LPS for 20 and 40 min. STAT1 serine phosphorylation was measured using Western blotting as described above. As shown in Fig. 2A, STAT1 serine phosphorylation was strongly induced in the C3H/OuJ macrophages. In contrast, no induction of STAT1 Ser-727 phosphorylation was observed in the TLR4 mutant macrophages. One of our published studies (20) has shown that TLR4 was also necessary for LPS-induced STAT1 tyrosine phosphorylation. In parallel studies, Pam3Cys was found to induce rapid STAT1 serine phosphorylation in macrophages from normal C57BL/6 mice but not from TLR2/ mice (Fig. 2B). Together, these findings demonstrate that TLRs 2 and 4 are necessary for STAT1 activation by Pam3Cys and E. coli LPS, respectively. Lastly, STAT1 serine phosphorylation could still be induced rapidly in the TLR4 mutant C3H/HeJ macrophages by the TLR2 agonist Pam3Cys and in TLR2-deficient macrophages by the TLR4 agonist E. coli LPS (data not shown).



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FIG. 2.
Induction of STAT1 serine phosphorylation in wild-type and TLR-deficient macrophages. A, peritoneal macrophages were isolated from C3H/OuJ control and TLR4 mutant C3H/HeJ mice as described under "Experimental Procedures." 3 days later, macrophages (3 x 106 cells in 60-mm dishes) were stimulated with E. coli LPS (100 ng/ml) for various times as indicated. Cell lysates were prepared, fractionated by SDS-PAGE, and then analyzed by Western blotting using an antibody against phospho-Ser-727 STAT1 (P-S(727)). Membranes were then stripped and re-probed using an antibody against total STAT1 as control for equal loading. RAW264.7 cells were also stimulated with IFN-{gamma} (200 units/ml, 4 h) as a positive control for phosphorylation of STAT1 on Ser-727. B, peritoneal macrophages from wild-type C57BL/6 and TLR2/ mice were prepared as described under "Experimental Procedures." After 3 days in culture (3 x 106 cells in 60-mm dishes), cells were stimulated with Pam3Cys (1 µg/ml) for the indicated times. STAT1 Ser-727 phosphorylation was then measured by Western blotting. Total STAT1 levels were measured as a control for equal loading. Lysates prepared from IFN-{gamma}-treated RAW264.7 cells were used as a positive control for STAT1 serine phosphorylation.

 

PI3K Mediates TLR-induced STAT1 Tyrosine Phosphorylation but Not Serine Phosphorylation—A previous study (24) has reported that serine phosphorylation of STAT1 could be mediated by PI3K in IFN-{gamma}-stimulated fibroblasts. We subsequently sought to determine whether STAT1 Ser-727 phosphorylation induced via TLR2 and TLR4 was also mediated by PI3K. To test this possibility, RAW264.7 macrophages were stimulated with E. coli LPS in the presence and absence of the specific PI3K inhibitor LY294002. As shown in Fig. 3, STAT1 serine phosphorylation was not inhibited by LY294002, demonstrating that this pathway is not dependent on PI3K. In contrast, both STAT1 tyrosine phosphorylation and phosphorylation of the PI3K-dependent kinase Akt were inhibited by LY294002 in a dose-dependent manner in LPS-stimulated RAW264.7 cells (Fig. 3B). As shown in Fig. 3C, STAT1 serine phosphorylation induced by Pam3Cys was not inhibited by LY294002, demonstrating that TLR2-dependent serine phosphorylation is not dependent on PI3K. Thus, PI3K mediates STAT1 tyrosine phosphorylation, activated via TLR4, but does not mediate serine phosphorylation in macrophages activated by engagement of TLR2 and TLR4.



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FIG. 3.
PI3K mediates TLR-dependent STAT1 tyrosine, but not serine, phosphorylation. RAW264.7 macrophages (3 x 106 cells) were plated in 60-mm dishes 1 day prior to the experiment. Cells were pretreated with various concentrations of LY294002 for 2 h and then stimulated with E. coli LPS (100 ng/ml) for 2 h to measure STAT1 Tyr-701 phosphorylation (A) or for 20 min to detect STAT1 Ser-727 phosphorylation (B). To verify that LY294002 was capable of suppressing PI3K activity, Akt Ser-473 phosphorylation was also measured from the same cell lysates used to detect STAT1 Ser-727 phosphorylation in B. Total STAT1 levels were measured as a control for equal loading. RAW264.7 cells were also treated with IFN-{gamma} (200 units/ml) for 4 h as a positive control of the phosphorylation of STAT1. C, RAW264.7 macrophages were stimulated with Pam3Cys (1 µg/ml) for 20 min in the presence of different concentrations of LY294002 as described above. STAT1 Ser-727 phosphorylation was measured by Western blotting. Akt Ser-473 phosphorylation was also measured by Western blotting using the same cell lysates to confirm that LY294002 blocked PI3K activity. Lysates prepared from RAW264.7 cells were treated with IFN-{gamma} (200 units/ml) for 4 h and were used as a positive control, and total STAT1 levels were used as a control for equal loading.

 

PI3K Does Not Mediate Induction of IFN-{beta} Gene Expression by E. coli LPS—IFN-{beta} has been shown previously (20) to mediate LPS-induced STAT1 tyrosine phosphorylation in macrophages. The finding that LY294002 could inhibit LPS-induced STAT1 tyrosine phosphorylation suggested that PI3K might be necessary for induction of IFN-{beta} expression in LPS-stimulated macrophages. To determine whether PI3K plays a role in LPS-induced IFN-{beta} production, RAW264.7 cells were stimulated with E. coli LPS in the presence and absence of LY294002. Total RNA was then isolated from the cells 2 h later, and IFN-{beta} mRNA levels were measured using semi-quantitative reverse transcriptase PCR. As shown in Fig. 4A, LY294002 did not inhibit LPS-induced endogenous IFN-{beta} mRNA expression. A second experimental approach was then used to confirm that PI3K does not mediate the activation of IFN-{beta} gene expression. RAW264.7 cells were co-transfected with an expression plasmid that encodes a constitutively active TLR4 mutant (TLR4-CA) and an IFN-{beta}-luciferase reporter plasmid. In some cases, cells were also transfected with an expression plasmid encoding a dominant-negative (kinase-dead) mutant of the p85{alpha} regulatory subunit. As shown in Fig. 4B, the dominant-negative p85{alpha} mutant failed to block TLR4-induced IFN-{beta} promoter activation in the transfected macrophages. The capacity of this dominant-negative p85{alpha} mutant to block activation of a PI3K-dependent promoter was confirmed in additional experiments using RAW264.7 cells co-transfected with an inducible nitric oxide synthase-luciferase reporter plasmid and the expression plasmid encoding the dominant-negative p85{alpha} mutant (data not shown). Together, these findings demonstrate that PI3K does not mediate activation of IFN-{beta} gene expression by LPS and TLR4.



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FIG. 4.
PI3K does not mediate TLR4-induced IFN-{beta} gene expression. A, RAW 264.7 macrophages were pretreated 30 min with either Me2SO (0.1% final concentration) or LY294002 (10 µM) and then stimulated for 3 h with E. coli LPS (100 ng/ml). Cells were then harvested, and total RNA was purified from the cell lysates. Four µg of total RNA was reverse-transcribed, and 200 ng of cDNA was amplified using semi-quantitative PCR to detect IFN-{beta} or {beta}-actin sequences as described previously (20). B, RAW264.7 cells were transiently co-transfected with a luciferase reporter plasmid under the control of the IFN-{beta} reporter plasmid (IFN-{beta}-luc; 1 µg), the constitutively active TLR4 expression plasmid (TLR4-CA; 1 µg), and a dominant-negative p85 (p85 (DN); 2 µg) in the indicated combinations. The HSP70-{beta}-galactosidase reporter construct was included as the internal control, and the empty vector was added as necessary to bring the total amount of plasmid DNA up to 4 µg for each transfection. Transfections were performed in triplicate, and a single representative experiment is shown. Data are reported as mean values ± S.E. (n = 3).

 

PI3K Does Not Mediate STAT1 Tyrosine Phosphorylation Induced by Exogenous IFN-{beta}The finding that PI3K did not mediate the induction of IFN-{beta} gene expression by LPS raised the alternative possibility that PI3K might be necessary for signaling via the type I IFN receptor (IFNAR). To test this possibility, the capacity of LY294002 to block STAT1 tyrosine phosphorylation induced by exogenous IFN-{beta} was evaluated. As shown in Fig. 5, exogenous IFN-{beta} rapidly induced STAT1 tyrosine phosphorylation, and LY294002 had a negligible effect on IFN-{beta}-induced STAT1 tyrosine phosphorylation. These findings demonstrate that PI3K does not mediate STAT1 activation via IFNAR signaling. Given the findings that PI3K was not necessary for either LPS-induced IFN-{beta} gene expression (Fig. 4) or IFNAR signaling (Fig. 5), these combined observations suggest that PI3K mediates LPS-induced IFN-{beta} secretion by macrophages (although not specifically tested in our studies). This possibility is consistent with the findings of Ohmori and Hamilton (25) who reported that LY294002 lowered IFN-{beta} secretion by LPS-stimulated RAW264.7 cells, compared with controls (25).



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FIG. 5.
PI3K does not mediate STAT1 serine phosphorylation induced by exogenous IFN-{beta} A, RAW264.7 macrophages (3 x 106 cells) were plated in 60-mm dishes 1 day prior to the experiment. Cells were then stimulated with recombinant murine IFN-{beta} (100 units/ml) for the indicated times. The cell lysates were fractionated by SDS-PAGE and analyzed by Western blotting using an antibody against phospho-Ser-727 STAT1. Membranes were then stripped and re-probed using an antibody against total STAT1 as control for equal loading. RAW264.7 cells were also treated with IFN-{gamma} (200 units/ml) for 4 h as a positive control of the phosphorylation of STAT1. B, cells were cultured as described above, pretreated with various concentrations of LY294002 for 2 h, and then stimulated with recombinant murine IFN-{beta} (100 units/ml) for 20 min to measure STAT1 Ser-727 phosphorylation. Total STAT1 levels were measured as a control for equal loading. Lysates prepared from RAW264.7 cells were treated with IFN-{gamma} (200 units/ml) for 4 h and were used as a positive control, and total STAT1 levels were used as a control for equal loading.

 

p38 MAP Kinase Mediates TLR-induced Serine Phosphorylation of STAT1—Although the identity of the protein kinase that phosphorylates STAT1 at serine residues in macrophages has not been established definitively (14), activation of serine STAT1 phosphorylation has been shown previously (27, 28) to be dependent on the p38 MAP kinase. To evaluate the role of p38 in TLR-induced STAT1 serine phosphorylation, RAW264.7 macrophages were stimulated with the TLR2 and TLR4 agonists Pam3Cys and E. coli LPS, respectively, in the presence and absence of the specific p38 inhibitor SB203580. A concentration of SB203580 was used (20 µM) that completely blocked TLR-dependent activation of p38 in RAW264.7 cells, without affecting viability of the cells (data not shown). Whole cell lysates were then analyzed by Western blotting using anti-phospho-p38 antibodies. As shown in Fig. 6, SB203580 treatment inhibited serine STAT1 phosphorylation induced by both Pam3Cys and E. coli LPS. These findings demonstrated that p38 was necessary for TLR-induced serine phosphorylation of STAT1.



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FIG. 6.
TLR-induced STAT1 serine phosphorylation requires p38 MAP kinase. Peritoneal macrophages (2 x 106 cells) from normal C3H/HeOuJ mice were prepared as described under "Experimental Procedures" and cultured in 6-well plates 3 days prior to the experiment. Cells were pre-treated with the selective p38 MAP kinase inhibitor SB203580 (20 µM) for 1 h as indicated. The cells were then stimulated with E. coli LPS (100 ng/ml) (A) or Pam3Cys (1 µg/ml) (B) as indicated. Cell lysates were prepared 20 min later, and Western blotting was used to measure STAT1 Ser-727 phosphorylation. Total STAT1 levels were also measured as a control for equal loading. Lysates prepared from IFN-{gamma}-treated RAW264.7 cells were used as a positive control for STAT1 activation. Pam, Pam3Cys; SB, SB203580.

 

p38 MAP Kinase Activation Is Not Sufficient to Induce STAT1 Serine Phosphorylation—Because both TLR proteins and the type I IL-1 receptor activate similar signal transduction pathways, including the activation of MAP kinases (3), the capacity of exogenous IL-1{beta} protein to activate STAT1 serine phosphorylation was also assessed. RAW264.7 macrophages were stimulated with recombinant murine IL-1{beta} (100 ng/ml) for 10 and 20 min. The activation of p38 and STAT1 was measured by Western blotting as described above. As shown in Fig. 7, p38 phosphorylation was rapidly induced in the macrophages following IL-1{beta} stimulation. In contrast, no induction of STAT1 serine phosphorylation was observed in the IL-1-stimulated macrophages, demonstrating that p38 activation is not sufficient for STAT1 activation. Together with the results shown in Fig. 6, these findings suggest that TLR engagement triggers the activation of a protein serine kinase that phosphorylates STAT1 in a p38-dependent manner. Both TLR agonists and IL-1{beta} are capable of activating p38, although only TLR agonists can activate STAT1 serine phosphorylation in macrophages. Thus, this protein serine kinase appears to distinguish the IL-1 from the TLR signaling pathways in these cells. Alternatively, IL-1{beta} may induce STAT1 serine phosphorylation while also activating a serine phosphatase that de-phosphorylates Ser-727.



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FIG. 7.
Activation of p38 MAP kinase is not sufficient to induce STAT1 serine phosphorylation. Peritoneal macrophages (2 x 106 cells) from normal C3H/HeOuJ mice were prepared as described under "Experimental Procedures" and cultured in 6-well plates 3 days prior to the experiment. The cells were then stimulated with exogenous recombinant murine IL-1{beta} (100 ng/ml) for various times as indicated. Cell lysates were prepared, and Western blotting was used to measure p38 and STAT1 Ser-727 phosphorylation. Total STAT1 levels were also measured as a control for equal loading. Lysates prepared from IFN-{gamma}-treated RAW264.7 cells were used as a positive control for STAT1 activation.

 

Role of PKC-{delta} in TLR-dependent Activation of STAT1 Serine Phosphorylation—Subsequent studies sought to determine the identity of the STAT1 serine kinase activated by engagement of TLR proteins. Several candidate kinases had been identified previously in macrophages, including isoforms of PKC. To evaluate the role of PKC isoforms in TLR-induced STAT1 serine phosphorylation, RAW264.7 macrophages were stimulated with the TLR2 and TLR4 agonists Pam3Cys and E. coli LPS, respectively, in the presence and absence of the pan-PKC-specific inhibitor bisindoylmaleamide or the PKC-{delta}-specific inhibitor rottlerin. Whole cell lysates were then analyzed by Western blotting. As shown in Fig. 8, both bisindoylmaleamide and rottlerin inhibited STAT1 Ser-727 phosphorylation induced by LPS but not Pam3Cys. Taken together, these data indicate that TLR2 and TLR4 engagement activates distinct STAT1 serine kinases and that the STAT1 serine kinase activated by E. coli LPS is a PKC family member, possibly PKC-{delta}.



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FIG. 8.
STAT1 serine phosphorylation induced via TLR4, but not via TLR2, is blocked by inhibitors of PKC-{delta} RAW264.7 macrophages (3 x 106 cells) were plated in 60-mm dishes 1 day prior to the experiment. Cells were pre-treated with the pan-PKC-specific inhibitor bisindoylmaleamide (20 µM)(A) or the PKC-{delta}-specific inhibitor rottlerin (20 µM) (B) for 1 h as indicated. The cells were then stimulated with E. coli LPS (100 ng/ml) or Pam3Cys (1 µg/ml) as indicated. Cell lysates were prepared 20 min later, and Western blotting was used to measure STAT1 Ser-727 phosphorylation. Total STAT1 levels were also measured as a control for equal loading. Pam, Pam3Cys; Rot, rottlerin; Bis, bisindoylmaleamide.

 

To obtain functional evidence of a role for PKC-{delta} in LPS-induced activation of STAT1, experiments were performed to determine whether a dominant-negative PKC-{delta} mutant could affect the trans-activation function of STAT1 in LPS-stimulated macrophages. RAW264.7 macrophages were transiently co-transfected with a luciferase reporter plasmid under the control of the STAT1-dependent IFN-{beta} promoter, with and without an expression plasmid encoding a dominant-negative (kinase-dead) PKC-{delta} mutant (19). As shown in Fig. 9A, E. coli LPS was a potent activator of the IFN-{beta} promoter in macrophages, and overexpression of the PKC-{delta} dominant-negative mutant in these cells resulted in a 36% average reduction in promoter activity. This is consistent with an inhibition of STAT1 serine phosphorylation, which would be expected to reduce (but not abolish) the trans-activation function of STAT1. The specificity of this PKC-{delta} dominant-negative mutant was confirmed by the finding that this mutant did not affect activation of the NF-{kappa}B-dependent COX2 promoter by LPS (Fig.9B). Together, these data provide further evidence of a role for PKC-{delta} in STAT1-dependent promoter activation by LPS.



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FIG. 9.
A PKC-{delta} dominant-negative mutant can partially block TLR4-induced activation of a STAT1-dependent promoter. RAW264.7 cells were transiently co-transfected with a luciferase reporter plasmid under the control of the IFN-{beta} reporter plasmid or murine COX2 reporter construct (2 µg) and either a dominant negative PKC-{delta} mutant expression plasmid (PKC-{delta} (DN); 2 µg) or empty vector (2 µg) in the indicated combinations. The HSP70-{beta}-galactosidase reporter construct (0.5 µg) was included as the internal control. Transfected cells were cultivated for a day and then stimulated with E. coli LPS (50 ng/ml) for 5 h as indicated. Transfections were performed in triplicate, and a single representative experiment is shown. Data are reported as mean values ± S.E. (n = 3).

 

Role of MyD88 in TLR-dependent Activation of STAT1 Serine Phosphorylation—The findings reported above demonstrated that STAT1 serine phosphorylation could be induced by engagement of TLR2 and TLR4 but not by IL-1{beta}. The adapter proteins MyD88 and TIRAP (Toll-interleukin 1 receptor domain-containing adapter protein) have been shown to mediate signal transduction via TLR2 and TLR4 (29, 30). In addition, a novel adapter protein, termed TRIF (Toll-interleukin 1 receptor domain-containing adapter inducing IFN-{beta}), may mediate signaling via TLR3 but not TLR2 (31). To assess the role of MyD88 in TLR-induced STAT1 serine phosphorylation, peritoneal macrophages were obtained from wild-type (C57BL/6) and MyD88/ mice. Cells were stimulated with E. coli LPS or Pam3Cys for various times as indicated. As shown in Fig. 10, both LPS and Pam3Cys induced rapid serine phosphorylation of STAT1 in wild-type macrophages. In MyD88-deficient macrophages, however, LPS also induced rapid STAT1 Ser-727 phosphorylation, whereas Pam3Cys did not. This demonstrates that activation of STAT1 serine phosphorylation via TLR2 is MyD88-dependent. Additional signaling components, such as TRIF, might provide an alternate pathway leading to STAT1 serine phosphorylation via TLR4. Together, these findings suggest a model in which STAT1 serine phosphorylation arises from two distinct signaling pathways. One pathway is TLR-specific and leads to the activation of a protein serine kinase, and the other pathway (via either MyD88 or TRIF) leads to the activation of p38 (Fig. 11). Neither pathway alone is sufficient to induce STAT1 Ser-727 phosphorylation.



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FIG. 10.
MyD88 is necessary for TLR2-induced, but not TLR4-induced, STAT1 serine phosphorylation. Peritoneal macrophages from the MyD88+/+ (C57BL/6) and MyD88/ mice were prepared as described under "Experimental Procedures" and cultured in 6-well plates 3 days prior to the experiment. The cells were then stimulated with LPS (100 ng/ml) (A) or with Pam3Cys (1 µg/ml) (B) for various times as indicated. Western blotting was used to measure STAT1 Ser-727 phosphorylation. Total STAT1 levels were also measured as a control for equal loading. Lysates prepared from IFN-{gamma}-treated RAW264.7 cells were used as a positive control for STAT1 activation.

 


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FIG. 11.
TLR-specific and p38-dependent activation of STAT1 Ser-727 phosphorylation. Shown is a model of signal transduction leading to STAT1 serine phosphorylation indicating the putative requirement of the p38 MAP kinase, PKC-{delta}, and a novel TLR2-specific pathway that leads to the activation of a non-PKC STAT1 serine protein kinase. Our data also support the possibility that either MyD88 or TIRAP can mediate TLR-specific activation of p38.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The objective of these studies was to characterize a novel signal transduction pathway initiated by engagement of TLR proteins. Many previous reports (6, 7) have documented the activation of MAP kinases and the transcription factors NF-{kappa}B and AP-1 by various members of the TLR family. Studies using genetically modified mice have demonstrated that activation of these signaling pathways is dependent on a variety of adapter proteins, such as MyD88, TIRAP, and TRIF. MyD88 and TI-RAP together appear to mediate signaling via TLR2 and TLR4 (29, 30), whereas TRIF participates in signaling via TLR3 (31). In the case of TLR4 signaling, additional adapter proteins may provide an alternate signaling pathway that can activate MAP kinases in a MyD88-independent manner (32). We reported previously (20) that TLR4-dependent signaling could activate cellular responses that are not activated by engagement of TLR2 and were not dependent on MyD88. Specifically, these responses include the induction of IFN-{beta} gene expression, activation of STAT1 tyrosine phosphorylation, and the induction of several STAT1-dependent genes (e.g. inducible nitric oxide synthase, IP-10, MCP-5). The current studies sought to characterize an additional response induced by TLR engagement, namely STAT1 serine phosphorylation, and to identify the factors necessary for this phosphorylation.

These studies revealed that STAT1 serine phosphorylation was rapidly induced following engagement of TLR2 and TLR4, as well as TLR9 (data not shown). These responses were dependent on TLR signaling as shown by the unresponsiveness of macrophages from TLR2/ or TLR4 mutant (C3H/HeJ) mice to the TLR agonists Pam3Cys and E. coli LPS, respectively. In contrast to STAT1 serine phosphorylation, STAT1 tyrosine phosphorylation was induced more slowly by E. coli LPS and not at all by Pam3Cys. In the case of LPS-induced STAT1 tyrosine phosphorylation, this response was shown previously (20) to be downstream of TLR4-induced IFN-{beta} production, IF-NAR engagement, and Jak/Tyk kinase activation. The inability of Pam3Cys to induce STAT1 tyrosine phosphorylation was because of the inability of this TLR2 agonist to induce IFN-{beta} production. Although a role for PI3K in STAT1 serine phosphorylation induced by E. coli LPS or Pam3Cys could not be demonstrated, the PI3K inhibitor LY294002 blocked STAT1 tyrosine phosphorylation induced by E. coli LPS. Additional experiments revealed that PI3K was not necessary for LPS-induced IFN-{beta} expression or for IFNAR signaling in response to exogenous IFN-{beta}. Therefore, these data support the possibility that PI3K mediates IFN-{beta} secretion by LPS-activated macrophages, a possibility suggested previously by Ohmori and Hamilton (25). Two published studies (27, 28) have reported that LPS-induced STAT1 serine phosphorylation was dependent on the p38 MAP kinase. These studies were confirmed and extended by showing that the p38 inhibitor SB203580 could also block Pam3Cys-induced STAT1 serine phosphorylation. Together with the finding that p38 activation by exogenous IL-1{beta} protein could not activate STAT1 serine phosphorylation in macrophages, our findings demonstrate that p38 is necessary, but not sufficient, for TLR-induced STAT1 activation.

Because the type I IL-1 receptor signals via MyD88 (33, 34), our data also suggest that MyD88 signaling is not sufficient for activation of STAT1 serine phosphorylation and that this response is mediated via a novel TLR-associated signaling pathway. The role of MyD88 in TLR-induced STAT1 serine phosphorylation was assessed directly using macrophages from MyD88/ mice. These studies revealed that MyD88 was necessary for TLR2-dependent activation of STAT1 but not for TLR4-dependent STAT1 activation. One likely explanation for this difference comes from the potential for the TLR4-specific adapter protein TRIF to activate p38, a kinase that is necessary for STAT1 serine phosphorylation. TRIF may activate MAP kinases in a MyD88-independent manner (16), thus providing a means to activate p38 in LPS-stimulated MyD88/ macrophages. Because TRIF does not mediate TLR2 activation by Pam3Cys, the TLR2 signaling pathway is solely dependent on MyD88 for activation of p38. This possibility is consistent with our finding that Pam3Cys failed to activate STAT1 serine phosphorylation in the MyD88/ macrophages. Although the existence of a MyD88-independent pathway leading to MAP kinase activation via TLR4 has been demonstrated previously, the specific adapter protein that mediates this pathway has not been identified definitively. Whether this adapter protein is TRIF or another novel factor remains to be determined.

Our studies also attempted to shed light on the identity of the STAT1 serine kinase activated by TLR engagement in macrophages. A previous report (28) has shown that p38 kinase itself only weakly phosphorylates STAT1 in vitro. More recently, additional kinases, including PKC-{delta}, PI3K, and calcium/calmodulin-dependent kinase II (24, 26, 35), have been shown to directly or indirectly mediate STAT1 serine phosphorylation in response to IFN signaling. Two pharmacological inhibitors of PKC were used to demonstrate a role for PKC isoforms in STAT1 serine phosphorylation induced via TLR4. Furthermore, the finding that rottlerin, a specific inhibitor of PKC-{delta}, could block LPS-induced STAT1 serine phosphorylation suggests a role for this particular PKC isoform. Consistent with this conclusion is the finding that a dominant-negative PKC-{delta} mutant partially blocking activation of a STAT1-dependent promoter by LPS. Unexpectedly, STAT1 serine phosphorylation in macrophages activated using Pam3Cys was not blocked by either rottlerin or by the pan-PKC inhibitor bisindoylmaleamide. Thus, PKC isoforms do not appear to play a role in STAT1 serine phosphorylation induced via TLR2. The identity of this additional serine kinase, and a reason for the existence of two distinct p38-dependent pathways leading to STAT1 Ser-727 phosphorylation, remain to be determined.

In summary, these findings demonstrate that distinct signal transduction pathways regulate TLR-dependent STAT1 serine and tyrosine phosphorylation in macrophages. This conclusion is consistent with previous studies performed using fibroblasts (13, 24) and macrophages (28). Tyrosine phosphorylation of STAT1 is indirectly mediated by the production of endogenous type I IFN, particularly IFN-{beta}, in LPS-stimulated macrophages (20). We have extended these earlier studies by showing that PI3K activation is necessary for STAT1 tyrosine phosphorylation in LPS-stimulated macrophages. Because PI3K does not appear to be necessary for the induction of IFN-{beta} gene expression, or signaling via the IFNAR, PI3K is likely to mediate the translation of IFN-{beta} mRNA and/or the secretion of newly synthesized IFN-{beta} protein. In contrast, STAT1 serine phosphorylation was clearly independent of PI3K in macrophages. Moreover, TLR-induced STAT1 serine phosphorylation was found to be dependent on both p38 and an additional STAT1 serine kinase, as discussed above. Thus, the mechanisms that regulate the DNA binding and trans-activation functions of STAT1 in macrophages, via phosphorylation of tyrosine and serine residues, respectively, are highly complex and differ somewhat from similar mechanisms that have been described previously in fibroblasts. The delineation of macrophage-specific mechanisms that regulate STAT1 serine phosphorylation will be the subject of future studies.


    FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants AI42733 (to M. J. F.) and AI18797 (to S. N. V.). 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. Back

To whom correspondence should be addressed: Dept. of Medicine, University of Maryland School of Medicine, MSTF-800, 685 W. Baltimore St., Baltimore, MD 21201-1192. Tel.: 410-706-0159; Fax: 410-706-8162; E-mail: mfenton{at}medicine.umaryland.edu.

1 The abbreviations used are: TLR, toll-like receptor; IFN, interferon; Pam3Cys, S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-Lys4-OH trihydrochloride; PI3K, phosphatidylinositol 3'-kinase; LPS, lipopolysaccharide; STAT, signal transducers and activators of transcription; IL, interleukin; MAP, mitogen-activated protein; PKC, protein kinase C. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Myriam Armant, Dr. Kurt Heldwein, and Sara Heiny for assistance with breeding and genotyping of the TLR2/ and MyD88/ mice, as well as the isolation of murine peritoneal macrophages.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Medzhitov, R., Preston-Hurlburt, P., and Janeway, C. A., Jr. (1997) Nature 388, 394–397[CrossRef][Medline] [Order article via Infotrieve]
  2. Poltorak, A., He, X., Smirnova, I., Liu, M. Y., Huffel, C. V., Du, X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M., Ricciardi-Castagnoli, P., Layton, B., and Beutler, B. (1998) Science 282, 2085–2088[Abstract/Free Full Text]
  3. Underhill, D. M., and Ozinsky, A. (2002) Curr. Opin. Immunol. 14, 103–110[CrossRef][Medline] [Order article via Infotrieve]
  4. Akira, S., Takeda, K., and Kaisho, T. (2001) Nat. Immunol. 2, 675–680[CrossRef][Medline] [Order article via Infotrieve]
  5. Re, F., and Strominger, J. L. (2001) J. Biol. Chem. 276, 37692–37699[Abstract/Free Full Text]
  6. Rhee, S. H., and Hwang, D. (2000) J. Biol. Chem. 275, 34035–34040[Abstract/Free Full Text]
  7. Kopp, E., Medzhitov, R., Carothers, J., Xiao, C., Douglas, I., Janeway, C. A., and Ghosh, S. (1999) Genes Dev. 13, 2059–2071[Abstract/Free Full Text]
  8. Herrera-Velit, P., and Reiner, N. E. (1996) J. Immunol. 156, 1157–1165[Abstract]
  9. Weinstein, S. L., Finn, A. J., Dave, S. H., Meng, F., Lowell, C. A., Sanghera, J. S., and DeFranco, A. L. (2000) J. Leukocyte Biol. 67, 405–414[Abstract]
  10. Darnell, J. E., Jr., Kerr, I. M., and Stark, G. R. (1994) Science 264, 1415–1421[Medline] [Order article via Infotrieve]
  11. Novick, D., Cohen, B., and Rubinstein, M. (1994) Cell 77, 391–400[Medline] [Order article via Infotrieve]
  12. Stark, G. R., Kerr, I. M., Williams, B. R., Silverman, R. H., and Schreiber, R. D. (1998) Annu. Rev. Biochem. 67, 227–264[CrossRef][Medline] [Order article via Infotrieve]
  13. Zhu, X., Wen, Z., Xu, L. Z., and Darnell, J. E., Jr. (1997) Mol. Cell. Biol. 17, 6618–6623[Abstract]
  14. Decker, T., and Kovarik, P. (2000) Oncogene 19, 2628–2637[CrossRef][Medline] [Order article via Infotrieve]
  15. Wen, Z., Zhong, Z., and Darnell, J. E., Jr. (1995) Cell 82, 241–250[Medline] [Order article via Infotrieve]
  16. Takeuchi, O., Hoshino, K., Kawai, T., Sanjo, H., Takada, H., Ogawa, T., Takeda, K., and Akira, S. (1999) Immunity 11, 443–451[Medline] [Order article via Infotrieve]
  17. Means, T. K., Lien, E., Yoshimura, A., Wang, S., Golenbock, D. T., and Fenton, M. J. (1999) J. Immunol. 163, 6748–6755[Abstract/Free Full Text]
  18. Wennstrom, S., and Downward, J. (1999) Mol. Cell. Biol. 19, 4279–4288[Abstract/Free Full Text]
  19. Murakami, M., Horowitz, A., Tang, S., Ware, J. A., and Simons, M. (2002) J. Biol. Chem. 277, 20367–20371[Abstract/Free Full Text]
  20. Toshchakov, V., Jones, B. W., Perera, P.-Y., Thomas, K., Cody, M. J., Zhang, S., Williams, B. R. G., Major, J., Hamilton, T. A., Fenton, M. J., and Vogel, S. N. (2002) Nat. Immunol. 3, 392–398[CrossRef][Medline] [Order article via Infotrieve]
  21. Hirschfeld, M., Ma, Y., Weis, J. H., Vogel, S. N., and Weis, J. J. (2000) J. Immunol. 165, 618–622[Abstract/Free Full Text]
  22. Brightbill, H. D., Libraty, D. H., Krutzik, S. R., Yang, R. B., Belisle, J. T., Bleharski, J. R., Maitland, M., Norgard, M. V., Plevy, S. E., Smale, S. T., Brennan, P. J., Bloom, B. R., Godowski, P. J., and Modlin, R. L. (1999) Science 285, 732–736[Abstract/Free Full Text]
  23. Laemmli, U. K. (1970) Nature 227, 680–685[Medline] [Order article via Infotrieve]
  24. Nguyen, H., Ramana, C. V., Bayes, J., and Stark, G. R. (2001) J. Biol. Chem. 276, 33361–33368[Abstract/Free Full Text]
  25. Ohmori, Y., and Hamilton, T. A. (2001) J. Leukocyte Biol. 69, 598–604[Abstract/Free Full Text]
  26. Nair, J. S., DaFonseca, C. J., Tjernberg, A., Sun, W., Darnell, J. E., Jr., Chait, B. T., and Zhang, J. J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5971–5976[Abstract/Free Full Text]
  27. Goh, K. C., Haque, S. J., and Williams, B. R. (1999) EMBO J. 18, 5601–5608[Abstract/Free Full Text]
  28. Kovarik, P., Stoiber, D., Eyers, P. A., Menghini, R., Neininger, A., Gaestel, M., Cohen, P., and Decker, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 13956–13961[Abstract/Free Full Text]
  29. Horng, T., Barton, G. M., Flavell, R. A., and Medzhitov, R. (2002) Nature 420, 329–333[CrossRef][Medline] [Order article via Infotrieve]
  30. Yamamoto, M., Sato, S., Hemmi, H., Sanjo, H., Uematsu, S., Kaisho, T., Hoshino, K., Takeuchi, O., Kobayashi, M., Fujita, T., Takeda, K., and Akira, S. (2002) Nature 420, 324–329[CrossRef][Medline] [Order article via Infotrieve]
  31. Yamamoto, M., Sato, S., Mori, K., Hoshino, K., Takeuchi, O., Takeda, K., and Akira, S. (2002) J. Immunol. 169, 6668–6672[Abstract/Free Full Text]
  32. Kawai, T., Adachi, O., Ogawa, T., Takeda, K., and Akira, S. (1999) Immunity 11, 11–22[Medline] [Order article via Infotrieve]
  33. Burns, K., Martinon, F., Esslinger C., Pahl, H., Schneider, P., Bodmer, J. L., Di Marco, F., French, L., and Tschopp, J. (1998) J. Biol. Chem. 273, 12203–12209[Abstract/Free Full Text]
  34. Adachi, O., Kawai, T., Takeda, K., Matsumoto, M., Tsutsui, H., Sakagami, M., Nakanishi, K., and Akira, S. (1998) Immunity 9, 143–150[Medline] [Order article via Infotrieve]
  35. Uddin, S., Sassano, A., Deb, D. K., Verma, A., Majchrzak, B., Rahman, A., Malik, A. B., Fish, E. N., and Platanias, L. C. (2002) J. Biol. Chem. 277, 14408–14416[Abstract/Free Full Text]