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

2003; Elsevier BV; Volume: 278; Issue: 25 Linguagem: Inglês

10.1074/jbc.m208633200

ISSN

1083-351X

Autores

Sang Hoon Rhee, Bryan W. Jones, Vladimir Y. Toshchakov, Stefanie N. Vogel, Matthew J. Fenton,

Tópico(s)

NF-κB Signaling Pathways

Resumo

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-δ. 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-δ, and a novel TLR2-specific signaling pathway appear to be necessary to induce Ser-727 STAT1 phosphorylation. 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-δ. 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-δ, and a novel TLR2-specific signaling pathway appear to be necessary to induce Ser-727 STAT1 phosphorylation. Mammalian Toll-like receptors (TLRs) 1The 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. are type I transmembrane receptors that are composed of an extracellular leucine-rich repeat domain and a highly conserved cytoplasmic Toll/IL-1R domain (1Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4528) Google Scholar). 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) (2Poltorak 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. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6578) Google Scholar). A variety of diverse chemical structures has been identified for most of the ten known TLR proteins (reviewed in Ref. 3Underhill D.M. Ozinsky A. Curr. Opin. Immunol. 2002; 14: 103-110Crossref PubMed Scopus (606) Google Scholar). 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 (4Akira S. Takeda K. Kaisho T. Nat. Immunol. 2001; 2: 675-680Crossref PubMed Scopus (4021) Google Scholar, 5Re F. Strominger J.L. J. Biol. Chem. 2001; 276: 37692-37699Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar). TLR agonists are known to activate multiple signal transduction pathways simultaneously in target cells. These signaling events include activation of the transcription factors NF-κB and AP-1, the MAP kinases, protein kinase C isoforms, and the lipid kinase phosphatidylinositol 3′-kinase (PI3K) (6Rhee S.H. Hwang D. J. Biol. Chem. 2000; 275: 34035-34040Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 7Kopp E. Medzhitov R. Carothers J. Xiao C. Douglas I. Janeway C.A. Ghosh S. Genes Dev. 1999; 13: 2059-2071Crossref PubMed Scopus (277) Google Scholar, 8Herrera-Velit P. Reiner N.E. J. Immunol. 1996; 156: 1157-1165PubMed Google Scholar, 9Weinstein S.L. Finn A.J. Dave S.H. Meng F. Lowell C.A. Sanghera J.S. DeFranco A.L. J. Leukocyte Biol. 2000; 67: 405-414Crossref PubMed Scopus (100) Google Scholar). 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-α/β) 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 (10Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5167) Google Scholar, 11Novick D. Cohen B. Rubinstein M. Cell. 1994; 77: 391-400Abstract Full Text PDF PubMed Scopus (596) Google Scholar). IFN-α/β 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 (10Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5167) Google Scholar, 12Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3408) Google Scholar). Phosphorylation of Tyr-701 alone is sufficient to generate STAT multimers that possess DNA binding activity (15Wen Z. Zhong Z. Darnell Jr., J.E. Cell. 1995; 82: 241-250Abstract Full Text PDF PubMed Scopus (1770) Google Scholar), although phosphorylation of Ser-727 is required for maximal transcriptional activity of STAT1 (reviewed in Ref. 14Decker T. Kovarik P. Oncogene. 2000; 19: 2628-2637Crossref PubMed Scopus (722) Google Scholar). 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 (14Decker T. Kovarik P. Oncogene. 2000; 19: 2628-2637Crossref PubMed Scopus (722) Google Scholar). 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-δ 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. 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 (16Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2830) Google Scholar). 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 (17Means T.K. Lien E. Yoshimura A. Wang S. Golenbock D.T. Fenton M.J. J. Immunol. 1999; 163: 6748-6755Crossref PubMed Google Scholar). 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 (6Rhee S.H. Hwang D. J. Biol. Chem. 2000; 275: 34035-34040Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). The dominant-negative p85 expression plasmid was kindly provided by Dr. Julian Downward (Imperial Cancer Research Fund, London, United Kingdom) and was described previously (18Wennstrom S. Downward J. Mol. Cell. Biol. 1999; 19: 4279-4288Crossref PubMed Scopus (255) Google Scholar). The PKC-δ dominant-negative expression plasmid was provided by Dr. Michael Simons (Dartmouth Medical School) and was described previously (19Murakami M. Horowitz A. Tang S. Ware J.A. Simons M. J. Biol. Chem. 2002; 277: 20367-20371Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The human IFN-β promoter luciferase reporter plasmid was provided by Dr. John Hiscott (McGill University, Montreal, Quebec, Canada) and was described previously (20Toshchakov 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. Vogel S.N. Nat. Immunol. 2002; 3: 392-398Crossref PubMed Scopus (694) Google Scholar). The murine COX2 promoter luciferase reporter plasmid was provided by Dr. Daniel Hwang (University of California, Davis, CA) and was also described previously (6Rhee S.H. Hwang D. J. Biol. Chem. 2000; 275: 34035-34040Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). 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. (21Hirschfeld M. Ma Y. Weis J.H. Vogel S.N. Weis J.J. J. Immunol. 2000; 165: 618-622Crossref PubMed Scopus (985) Google Scholar). 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-γ was purchased from R & D Systems (Minneapolis, MN), and recombinant murine IL-1β 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 × 106 cells/well) and transfected with the appropriate plasmid DNA, including a β-galactosidase expression plasmid (HSP70-β-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 β-galactosidase activity as described previously (6Rhee S.H. Hwang D. J. Biol. Chem. 2000; 275: 34035-34040Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 22Brightbill 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. Modlin R.L. Science. 1999; 285: 732-736Crossref PubMed Scopus (1421) Google Scholar). 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 × 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 (23Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (214196) Google Scholar). 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 (6Rhee S.H. Hwang D. J. Biol. Chem. 2000; 275: 34035-34040Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). 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 (20Toshchakov 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. Vogel S.N. Nat. Immunol. 2002; 3: 392-398Crossref PubMed Scopus (694) Google Scholar). The oligonucleotide primers used for amplification of the murine IFN-β PCR product were 5′-TCCAAGAAAGGACGAACATTCG-3′ and 5′-TGAGGACATCTCCCACGTCAA-3′ (annealing temperature, 55 °C). 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-κB and AP-1 in macrophages (6Rhee S.H. Hwang D. J. Biol. Chem. 2000; 275: 34035-34040Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 7Kopp E. Medzhitov R. Carothers J. Xiao C. Douglas I. Janeway C.A. Ghosh S. Genes Dev. 1999; 13: 2059-2071Crossref PubMed Scopus (277) Google Scholar). 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 (20Toshchakov 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. Vogel S.N. Nat. Immunol. 2002; 3: 392-398Crossref PubMed Scopus (694) Google Scholar) and is because of the inability of TLR2 agonists to induce IFN-β secretion and subsequent engagement of the type I IFN receptor. 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 STAT1α (92 kDa) and STAT1β (84 kDa). A single species of serine-phosphorylated STAT1 was observed, corresponding to STAT1α. The STAT1β splice variant lacks the C-terminal serine phosphorylation site present in STAT1α. In the figures, this serine-phosphorylated form of STAT1α will simply be referred to as P-S(727)STAT1. STAT1 tyrosine phosphorylation was first observed ∼2 h after LPS stimulation, as reported previously (20Toshchakov 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. Vogel S.N. Nat. Immunol. 2002; 3: 392-398Crossref PubMed Scopus (694) Google Scholar), 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 (20Toshchakov 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. Vogel S.N. Nat. Immunol. 2002; 3: 392-398Crossref PubMed Scopus (694) Google Scholar) 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). PI3K Mediates TLR-induced STAT1 Tyrosine Phosphorylation but Not Serine Phosphorylation—A previous study (24Nguyen H. Ramana C.V. Bayes J. Stark G.R. J. Biol. Chem. 2001; 276: 33361-33368Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) has reported that serine phosphorylation of STAT1 could be mediated by PI3K in IFN-γ-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. PI3K Does Not Mediate Induction of IFN-β Gene Expression by E. coli LPS—IFN-β has been shown previously (20Toshchakov 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. Vogel S.N. Nat. Immunol. 2002; 3: 392-398Crossref PubMed Scopus (694) Google Scholar) 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-β expression in LPS-stimulated macrophages. To determine whether PI3K plays a role in LPS-induced IFN-β 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-β mRNA levels were measured using semi-quantitative reverse transcriptase PCR. As shown in Fig. 4A, LY294002 did not inhibit LPS-induced endogenous IFN-β mRNA expression. A second experimental approach was then used to confirm that PI3K does not mediate the activation of IFN-β gene expression. RAW264.7 cells were co-transfected with an expression plasmid that encodes a constitutively active TLR4 mutant (TLR4-CA) and an IFN-β-luciferase reporter plasmid. In some cases, cells were also transfected with an expression plasmid encoding a dominant-negative (kinase-dead) mutant of the p85α regulatory subunit. As shown in Fig. 4B, the dominant-negative p85α mutant failed to block TLR4-induced IFN-β promoter activation in the transfected macrophages. The capacity of this dominant-negative p85α 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 p85α mutant (data not shown). Together, these findings demonstrate that PI3K does not mediate activation of IFN-β gene expression by LPS and TLR4. PI3K Does Not Mediate STAT1 Tyrosine Phosphorylation Induced by Exogenous IFN-β—The finding that PI3K did not mediate the induction of IFN-β 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-β was evaluated. As shown in Fig. 5, exogenous IFN-β rapidly induced STAT1 tyrosine phosphorylation, and LY294002 had a negligible effect on IFN-β-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-β gene expression (Fig. 4) or IFNAR signaling (Fig. 5), these combined observations suggest that PI3K mediates LPS-induced IFN-β secretion by macrophages (although not specifically tested in our studies). This possibility is consistent with the findings of Ohmori and Hamilton (25Ohmori Y. Hamilton T.A. J. Leukocyte Biol. 2001; 69: 598-604PubMed Google Scholar) who reported that LY294002 lowered IFN-β secretion by LPS-stimulated RAW264.7 cells, compared with controls (25Ohmori Y. Hamilton T.A. J. Leukocyte Biol. 2001; 69: 598-604PubMed Google Scholar). 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 (14Decker T. Kovarik P. Oncogene. 2000; 19: 2628-2637Crossref PubMed Scopus (722) Google Scholar), activation of serine STAT1 phosphorylation has been shown previously (27Goh K.C. Haque S.J. Williams B.R. EMBO J. 1999; 18: 5601-5608Crossref PubMed Scopus (329) Google Scholar, 28Kovarik P. Stoiber D. Eyers P.A. Menghini R. Neininger A. Gaestel M. Cohen P. Decker T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13956-13961Crossref PubMed Scopus (230) Google Scholar) 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. 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 (3Underhill D.M. Ozinsky A. Curr. Opin. Immunol. 2002; 14: 103-110Crossref PubMed Scopus (606) Google Scholar), the capacity of exogenous IL-1β protein to activate STAT1 serine phosphorylation was also assessed. RAW264.7 macrophages were stimulated with recombinant murine IL-1β (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-1β 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-1β 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-1β may induce STAT1 serine phosphorylation while also activating a serine phosphatase that de-phosphorylates Ser-727. Role of PKC-δ 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-δ-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-δ. To obtain functional evidence of a role for PKC-δ in LPS-induced activation of STAT1, experiments were performed to determine whether a dominant-negative PKC-δ 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-β promoter, with and without an expression plasmid encoding a dominant-negative (kinase-dead) PKC-δ mutant (19Murakami M. Horowitz A. Tang S. Ware J.A. Simons M. J. Biol. Chem. 2002; 277: 20367-20371Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). As shown in Fig. 9A, E. coli LPS was a potent activator of the IFN-β promoter in macrophages, and overexpression of the PKC-δ 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-δ dominant-negative mutant was confirmed by the finding that this mutant did not affect activation of the NF-κB-dependent COX2 promoter by LPS (Fig.9B). Together, these data provide further evidence of a role for PKC-δ in STAT1-dependent promoter activation by LPS. 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-1β. 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 (29Horng T. Barton G.M. Flavell R.A. Medzhitov R. Nature. 2002; 420: 329-333Crossref PubMed Scopus (694) Google Scholar, 30Yamamoto M. Sato S. Hemmi H. Sanjo H. Uematsu S. Kaisho T. Hoshino K. Takeuchi O. Kobayashi M. Fujita T. Takeda K. Akira S. Nature. 2002; 420: 324-329Crossref PubMed Scopus (829) Google Scholar). In addition, a novel adapter protein, termed TRIF (Toll-interleukin 1 receptor domain-containing adapter inducing IFN-β), may mediate signaling via TLR3 but not TLR2 (31Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. J. Immunol. 2002; 169: 6668-6672Crossref PubMed Scopus (1034) Google Scholar). 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.Fig. 11TLR-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-δ, 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.View Large Image Figure ViewerDownload (PPT) The objective of these studies was to characterize a novel signal transduction pathway initiated by engagement of TLR proteins. Many previous reports (6Rhee S.H. Hwang D. J. Biol. Chem. 2000; 275: 34035-34040Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 7Kopp E. Medzhitov R. Carothers J. Xiao C. Douglas I. Janeway C.A. Ghosh S. Genes Dev. 1999; 13: 2059-2071Crossref PubMed Scopus (277) Google Scholar) have documented the activation of MAP kinases and the transcription factors NF-κB 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 (29Horng T. Barton G.M. Flavell R.A. Medzhitov R. Nature. 2002; 420: 329-333Crossref PubMed Scopus (694) Google Scholar, 30Yamamoto M. Sato S. Hemmi H. Sanjo H. Uematsu S. Kaisho T. Hoshino K. Takeuchi O. Kobayashi M. Fujita T. Takeda K. Akira S. Nature. 2002; 420: 324-329Crossref PubMed Scopus (829) Google Scholar), whereas TRIF participates in signaling via TLR3 (31Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. J. Immunol. 2002; 169: 6668-6672Crossref PubMed Scopus (1034) Google Scholar). 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 (32Kawai T. Adachi O. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 11-22Abstract Full Text Full Text PDF PubMed Scopus (1748) Google Scholar). We reported previously (20Toshchakov 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. Vogel S.N. Nat. Immunol. 2002; 3: 392-398Crossref PubMed Scopus (694) Google Scholar) 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-β 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 (20Toshchakov 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. Vogel S.N. Nat. Immunol. 2002; 3: 392-398Crossref PubMed Scopus (694) Google Scholar) to be downstream of TLR4-induced IFN-β 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-β 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-β expression or for IFNAR signaling in response to exogenous IFN-β. Therefore, these data support the possibility that PI3K mediates IFN-β secretion by LPS-activated macrophages, a possibility suggested previously by Ohmori and Hamilton (25Ohmori Y. Hamilton T.A. J. Leukocyte Biol. 2001; 69: 598-604PubMed Google Scholar). Two published studies (27Goh K.C. Haque S.J. Williams B.R. EMBO J. 1999; 18: 5601-5608Crossref PubMed Scopus (329) Google Scholar, 28Kovarik P. Stoiber D. Eyers P.A. Menghini R. Neininger A. Gaestel M. Cohen P. Decker T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13956-13961Crossref PubMed Scopus (230) Google Scholar) 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-1β 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 (33Burns K. Martinon F. Esslinger C. Pahl H. Schneider P. Bodmer J.L. Di Marco F. French L. Tschopp J. J. Biol. Chem. 1998; 273: 12203-12209Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar, 34Adachi O. Kawai T. Takeda K. Matsumoto M. Tsutsui H. Sakagami M. Nakanishi K. Akira S. Immunity. 1998; 9: 143-150Abstract Full Text Full Text PDF PubMed Scopus (1744) Google Scholar), 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 (16Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2830) Google Scholar), 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 (28Kovarik P. Stoiber D. Eyers P.A. Menghini R. Neininger A. Gaestel M. Cohen P. Decker T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13956-13961Crossref PubMed Scopus (230) Google Scholar) has shown that p38 kinase itself only weakly phosphorylates STAT1 in vitro. More recently, additional kinases, including PKC-δ, PI3K, and calcium/calmodulin-dependent kinase II (24Nguyen H. Ramana C.V. Bayes J. Stark G.R. J. Biol. Chem. 2001; 276: 33361-33368Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 26Nair J.S. DaFonseca C.J. Tjernberg A. Sun W. Darnell Jr., J.E. Chait B.T. Zhang J.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5971-5976Crossref PubMed Scopus (102) Google Scholar, 35Uddin S. Sassano A. Deb D.K. Verma A. Majchrzak B. Rahman A. Malik A.B. Fish E.N. Platanias L.C. J. Biol. Chem. 2002; 277: 14408-14416Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), 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-δ, 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-δ 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 (13Zhu X. Wen Z. Xu L.Z. Darnell Jr., J.E. Mol. Cell. Biol. 1997; 17: 6618-6623Crossref PubMed Scopus (141) Google Scholar, 24Nguyen H. Ramana C.V. Bayes J. Stark G.R. J. Biol. Chem. 2001; 276: 33361-33368Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) and macrophages (28Kovarik P. Stoiber D. Eyers P.A. Menghini R. Neininger A. Gaestel M. Cohen P. Decker T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13956-13961Crossref PubMed Scopus (230) Google Scholar). Tyrosine phosphorylation of STAT1 is indirectly mediated by the production of endogenous type I IFN, particularly IFN-β, in LPS-stimulated macrophages (20Toshchakov 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. Vogel S.N. Nat. Immunol. 2002; 3: 392-398Crossref PubMed Scopus (694) Google Scholar). 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-β gene expression, or signaling via the IFNAR, PI3K is likely to mediate the translation of IFN-β mRNA and/or the secretion of newly synthesized IFN-β 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. 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.

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