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Glucocorticoids Inhibit IRF3 Phosphorylation in Response to Toll-like Receptor-3 and -4 by Targeting TBK1 Activation

2008; Elsevier BV; Volume: 283; Issue: 21 Linguagem: Inglês

10.1074/jbc.m709731200

ISSN

1083-351X

Autores

Claire E. McCoy, Susan Carpenter, Eva M. Pålsson‐McDermott, Linden J. Gearing, Luke O'neill,

Tópico(s)

Cancer-related molecular mechanisms research

Resumo

Phosphorylation of the transcription factor interferon regulatory factor 3 (IRF3) is essential for the induction of promoters which contain the interferon-stimulated response element (ISRE). IRF3 can be activated by Toll-like receptor 3 (TLR3) in response to the double-stranded RNA mimic poly(I-C) and by TLR4 in response to lipopolysaccharide (LPS). Here we have analyzed the effect of the glucocorticoid dexamethasone on this response. Dexamethasone inhibited the induction of the ISRE-dependent gene RANTES (regulated on activation normal T cell expressed and secreted) in both U373-CD14 cells and human peripheral blood mononuclear cells and also an ISRE luciferase construct, activated by either TLR3 or TLR4. It also inhibited increased phosphorylation of IRF3 in its N terminus in response to LPS and in its C terminus on Ser-396 in response to either poly(I-C) or LPS. Several dexamethasone-induced phosphatases were tested for possible involvement in these effects; MKP1 did not appear to be involved, although MKP2 and MKP5 both partially inhibited induction of the ISRE, pointing to their possible involvement in the effect of dexamethasone. Importantly, we found that dexamethasone could inhibit TBK1 kinase activity and TBK1 phosphorylation on Ser-172, both of which are required for IRF3 phosphorylation downstream of TLR3 and TLR4 stimulation. Our study, therefore, demonstrates that TBK1 is a target for dexamethasone, common to both TLR3 and TLR4 signaling. Phosphorylation of the transcription factor interferon regulatory factor 3 (IRF3) is essential for the induction of promoters which contain the interferon-stimulated response element (ISRE). IRF3 can be activated by Toll-like receptor 3 (TLR3) in response to the double-stranded RNA mimic poly(I-C) and by TLR4 in response to lipopolysaccharide (LPS). Here we have analyzed the effect of the glucocorticoid dexamethasone on this response. Dexamethasone inhibited the induction of the ISRE-dependent gene RANTES (regulated on activation normal T cell expressed and secreted) in both U373-CD14 cells and human peripheral blood mononuclear cells and also an ISRE luciferase construct, activated by either TLR3 or TLR4. It also inhibited increased phosphorylation of IRF3 in its N terminus in response to LPS and in its C terminus on Ser-396 in response to either poly(I-C) or LPS. Several dexamethasone-induced phosphatases were tested for possible involvement in these effects; MKP1 did not appear to be involved, although MKP2 and MKP5 both partially inhibited induction of the ISRE, pointing to their possible involvement in the effect of dexamethasone. Importantly, we found that dexamethasone could inhibit TBK1 kinase activity and TBK1 phosphorylation on Ser-172, both of which are required for IRF3 phosphorylation downstream of TLR3 and TLR4 stimulation. Our study, therefore, demonstrates that TBK1 is a target for dexamethasone, common to both TLR3 and TLR4 signaling. Interferon-regulatory factor 3 (IRF3) 2The abbreviations used are: IRF3, interferon-regulatory factor 3; TLR, Toll-like receptor; ISRE, interferon-stimulated response element; MKP1, MAP kinase phosphatase; RIG-I, retinoic acid-inducible gene I; IFN, interferon; LPS, lipopolysaccharide; RANTES, regulated on activation normal T cell expressed and secreted; GST, glutathione S-transferase; MAP, mitogen-activated protein; PBMC, peripheral blood mononuclear cells; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. 2The abbreviations used are: IRF3, interferon-regulatory factor 3; TLR, Toll-like receptor; ISRE, interferon-stimulated response element; MKP1, MAP kinase phosphatase; RIG-I, retinoic acid-inducible gene I; IFN, interferon; LPS, lipopolysaccharide; RANTES, regulated on activation normal T cell expressed and secreted; GST, glutathione S-transferase; MAP, mitogen-activated protein; PBMC, peripheral blood mononuclear cells; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. is a transcription factor that is activated through recognition of viral double-stranded RNA by receptors such as Toll-like receptor 3 (TLR3) or by intracellular receptors such as retinoic acid-inducible gene I (RIG-I) (1Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (755) Google Scholar, 2Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (688) Google Scholar, 3Weaver B.K. Kumar K.P. Reich N.C. Mol. Cell. Biol. 1998; 18: 1359-1368Crossref PubMed Scopus (297) Google Scholar, 4Servant M.J. ten Oever B. LePage C. Conti L. Gessani S. Julkunen I. Lin R. Hiscott J. J. Biol. Chem. 2001; 276: 355-363Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 5Iwamura T. Yoneyama M. Yamaguchi K. Suhara W. Mori W. Shiota K. Okabe Y. Namiki H. Fujita T. Genes Cells. 2001; 6: 375-388Crossref PubMed Scopus (236) Google Scholar). Recognition of bacterial components such as lipopolysaccharide (LPS) by TLR4 also leads to IRF3 activation (6Navarro L. David M. J. Biol. Chem. 1999; 274: 35535-35538Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 7Sakaguchi S. Negishi H. Asagiri M. Nakajima C. Mizutani T. Takaoka A. Honda K. Taniguchi T. Biochem. Biophys. Res. Commun. 2003; 306: 860-866Crossref PubMed Scopus (208) Google Scholar, 8Solis M. Romieu-Mourez R. Goubau D. Grandvaux N. Mesplede T. Julkunen I. Nardin A. Salcedo M. Hiscott J. Eur. J. Immunol. 2007; 37: 528-539Crossref PubMed Scopus (43) Google Scholar). In response to these stimuli, IRF3 becomes readily phosphorylated, resulting in IRF3 dimerization, association with co-factor cAMP-response element-binding protein (CREB)-binding protein, and subsequent translocation to the nucleus (1Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (755) Google Scholar, 9Servant M.J. Grandvaux N. tenOever B.R. Duguay D. Lin R. Hiscott J. J. Biol. Chem. 2003; 278: 9441-9447Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Entry to the nucleus allows IRF3 to bind to consensus DNA sequences known as the interferon (IFN)-stimulated response element (ISRE) found in the promoter regions of genes such as those encoding IFN-β, IFN-α1, CXC-chemokine ligand 10 (CXCL10), and RANTES (10Schafer S.L. Lin R. Moore P.A. Hiscott J. Pitha P.M. J. Biol. Chem. 1998; 273: 2714-2720Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 11Lin R. Heylbroeck C. Genin P. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1999; 19: 959-966Crossref PubMed Scopus (250) Google Scholar, 12Sing A. Merlin T. Knopf H.P. Nielsen P.J. Loppnow H. Galanos C. Freudenberg M.A. Infect. Immun. 2000; 68: 1600-1607Crossref PubMed Scopus (75) Google Scholar). These IRF3-dependent genes play an important role in both the anti-viral and anti-bacterial innate immune response (13Honda K. Taniguchi T. Nat. Rev. Immunol. 2006; 6: 644-658Crossref PubMed Scopus (1266) Google Scholar). Multiple phosphorylation sites have been identified on IRF3. Phosphorylation of Ser-396, which lies in a cluster of 5 serine/threonine residues (Ser-396, Ser-398, Ser-402, Thr-404, Ser-405) located in the C terminus is particularly important for IRF3 activation as mutation of this site alone to a phosphomimetic aspartic acid generates a constitutively active form of IRF3 which can strongly induce the ISRE promoter element of genes encoding IFN-β, IFN-α1, and RANTES (9Servant M.J. Grandvaux N. tenOever B.R. Duguay D. Lin R. Hiscott J. J. Biol. Chem. 2003; 278: 9441-9447Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Phosphorylation of this site is also critical for IRF3 dimerization and association with cAMP-response element-binding protein (CREB)-binding protein as well as nuclear translocation (1Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (755) Google Scholar, 9Servant M.J. Grandvaux N. tenOever B.R. Duguay D. Lin R. Hiscott J. J. Biol. Chem. 2003; 278: 9441-9447Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 14Clement J.F. Bibeau-Poirier A. Gravel S.P. Grandvaux N. Bonneil E. Thibault P. Meloche S. Servant M.J. J. Virol. 2008; 82: 3984-3996Crossref PubMed Scopus (71) Google Scholar). Ser-386, which lies proximal to the C-terminal cluster, also appears to be important as mutation of this residue to an alanine abolishes the ability of IRF3 to dimerize, a function that is critical for the translocation of IRF3 to the nucleus (15Mori M. Yoneyama M. Ito T. Takahashi K. Inagaki F. Fujita T. J. Biol. Chem. 2004; 279: 9698-9702Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). The role for this multitude of IRF3 phosphorylation sites has been clarified in a more recent study detailing that activation of IRF3 appears to be a sequential process of phosphorylation, where phosphorylation of Ser-396 occurs first followed by phosphorylation of Ser-404 and Ser-405, thereby priming IRF3 for phosphorylation on Ser-386, required for dimerization (14Clement J.F. Bibeau-Poirier A. Gravel S.P. Grandvaux N. Bonneil E. Thibault P. Meloche S. Servant M.J. J. Virol. 2008; 82: 3984-3996Crossref PubMed Scopus (71) Google Scholar). Each of these sites plays a slightly different but equally important role in overall IRF3 activation and transcriptional activation of IRF3-dependent genes. The same study also identified Ser-339 as another important residue that appears to share a redundant role with that of Ser-396 (14Clement J.F. Bibeau-Poirier A. Gravel S.P. Grandvaux N. Bonneil E. Thibault P. Meloche S. Servant M.J. J. Virol. 2008; 82: 3984-3996Crossref PubMed Scopus (71) Google Scholar). IRF3 can also be phosphorylated within its N terminus by stress inducers such as anisomycin, sorbitol, and DNA-damaging agents such as doxorubicin (4Servant M.J. ten Oever B. LePage C. Conti L. Gessani S. Julkunen I. Lin R. Hiscott J. J. Biol. Chem. 2001; 276: 355-363Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). LPS (but not double-stranded RNA) has also been shown to induce N-terminal phosphorylation (8Solis M. Romieu-Mourez R. Goubau D. Grandvaux N. Mesplede T. Julkunen I. Nardin A. Salcedo M. Hiscott J. Eur. J. Immunol. 2007; 37: 528-539Crossref PubMed Scopus (43) Google Scholar). TBK1 and IKKϵ are two serine/threonine kinases that have been shown to lie upstream of IRF3 and are required for phosphorylation of the C-terminal cluster, nuclear translocation and, activation of IRF3-dependent ISRE reporters (16Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (2068) Google Scholar, 17Sharma S. tenOever B.R. Grandvaux N. Zhou G.P. Lin R. Hiscott J. Science. 2003; 300: 1148-1151Crossref PubMed Scopus (1357) Google Scholar, 18McWhirter S.M. Fitzgerald K.A. Rosains J. Rowe D.C. Golenbock D.T. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 233-238Crossref PubMed Scopus (450) Google Scholar). TBK1–/– mouse embryonic fibroblasts were shown to be defective in IRF3 nuclear translocation and IFN-α1, IFN-β, and RANTES gene expression in response to viral infection (both Sendai and Newcastle disease virus), poly(I-C) (a double-stranded RNA mimic), and LPS stimulation (18McWhirter S.M. Fitzgerald K.A. Rosains J. Rowe D.C. Golenbock D.T. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 233-238Crossref PubMed Scopus (450) Google Scholar, 19Perry A.K. Chow E.K. Goodnough J.B. Yeh W.C. Cheng G. J. Exp. Med. 2004; 199: 1651-1658Crossref PubMed Scopus (310) Google Scholar, 20Hemmi H. Takeuchi O. Sato S. Yamamoto M. Kaisho T. Sanjo H. Kawai T. Hoshino K. Takeda K. Akira S. J. Exp. Med. 2004; 199: 1641-1650Crossref PubMed Scopus (461) Google Scholar). Solis et al. (8Solis M. Romieu-Mourez R. Goubau D. Grandvaux N. Mesplede T. Julkunen I. Nardin A. Salcedo M. Hiscott J. Eur. J. Immunol. 2007; 37: 528-539Crossref PubMed Scopus (43) Google Scholar) more recently showed that LPS could activate both TBK1 and IKKϵ in macrophages but with different kinetics, the effect on TBK1 being much more rapid and pre-dominant. Knockdown of both, however, with small interfering RNA inhibited IFN-β expression in macrophages stimulated with LPS (8Solis M. Romieu-Mourez R. Goubau D. Grandvaux N. Mesplede T. Julkunen I. Nardin A. Salcedo M. Hiscott J. Eur. J. Immunol. 2007; 37: 528-539Crossref PubMed Scopus (43) Google Scholar). Phosphorylation of IRF3 by TBK1, therefore, appears to be a common component of signaling pathways activated by RIG-I, TLR3, and TLR4. However, evidence has emerged to show that specificity can arise within these pathways. One important observation showed that the nuclear factor κB component p65 can form a complex with IRF3 downstream of TLR4 activation but not TLR3 (21Wietek C. Miggin S.M. Jefferies C.A. O'Neill L.A. J. Biol. Chem. 2003; 278: 50923-50931Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). This observation was supported in an elegant study by Ogawa et al. (22Ogawa S. Lozach J. Benner C. Pascual G. Tangirala R.K. Westin S. Hoffmann A. Subramaniam S. David M. Rosenfeld M.G. Glass C.K. Cell. 2005; 122: 707-721Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar), which showed that a subset of ISRE-dependent genes activated by LPS were inhibited by the glucocorticoid dexamethasone through the disruption of this p65/IRF3 complex. The same set of genes when induced by poly(I-C) were, however, insensitive to dexamethasone since p65 is not in the IRF3 complex when activated by this stimulus. In a more recent study, however, Reily et al. (23Reily M.M. Pantoja C. Hu X. Chinenov Y. Rogatsky I. EMBO J. 2006; 25: 108-117Crossref PubMed Scopus (128) Google Scholar) showed that dexamethasone could in fact inhibit the expression of certain IRF3-dependent genes such as RANTES, IFN-β, IP10, ISG15, and ISG56 downstream of TLR3 stimulation by poly(I-C). They showed that dexamethasone could disrupt the ability of a coactivator protein called glucocorticoid receptor-interacting protein to interact with IRF3, thereby suppressing IRF3-dependent genes (23Reily M.M. Pantoja C. Hu X. Chinenov Y. Rogatsky I. EMBO J. 2006; 25: 108-117Crossref PubMed Scopus (128) Google Scholar). The basis for this discrepancy with the study performed by Ogawa et al. (22Ogawa S. Lozach J. Benner C. Pascual G. Tangirala R.K. Westin S. Hoffmann A. Subramaniam S. David M. Rosenfeld M.G. Glass C.K. Cell. 2005; 122: 707-721Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar) is unclear. The fact that IRF3 is a common component to both the TLR4 and TLR3 pathways led us to examine the effect of glucocorticoids on IRF3 phosphorylation. In this study we have found that dexamethasone can inhibit the induction of an ISRE-dependent reporter gene and the IRF3-dependent gene encoding RANTES by both LPS and poly(I-C). Importantly, we suggest that this is due to a decrease of IRF3 phosphorylation due to the inhibition of TBK1 kinase activity. This demonstrates for the first time that TBK1 is a target for glucocorticoids downstream of TLR4 and TLR3 activation. Plasmids and Reagents—The ISRE luciferase plasmid was purchased from Clontech. pcDNA3.1 empty vector was from Invitrogen. pCMV-MKP1 wild-type and in-active MKP1C258S were kind gifts from Andrew Clark (Imperial College London). pEFr-FLAG PAC-1 expression plasmid was obtained from Steve Gerondakis (WEHI, Melbourne). pSG5-myc-tagged DUSP4–7, DUSP8, and DUSP10 plasmids were generous gifts from Stephen Keyse (Ninewells hospital, Dundee, Scotland). Constitutively active Calcineurin pEGFP CN-CaMIA was obtained from Young Jun Kang and Masato Kubo (RIKEN Yokohama Institute) (24Kang Y.J. Kusler B. Otsuka M. Hughes M. Suzuki N. Suzuki S. Yeh W.C. Akira S. Han J. Jones P.P. J. Immunol. 2007; 179: 4598-4607Crossref PubMed Scopus (83) Google Scholar). Wild-type GST-IRF3 (380–427), GST-IRF3 (5A), and GST-IRF3 (7A) were kind gifts from Katherine Fitzgerald (University of Massachusetts Medical School). pcDNA3-FLAG kinase dead TBK1 plasmid was obtained from Makoto Nakanishi (Nagoya City University). The following antibodies were used: total IRF3 (Santa Cruz Biotechnology, Inc.), phospho-Ser-396 IRF3, and phospho-p38 MAP kinase (Cell Signaling Technology Inc.), anti-TBK1 (Imgenex), and anti-β-actin (Sigma). Anti-NAK (TBK1) and anti-TBK1 (Ser-172) used for immunoprecipitation assays were from Abcam (Cambridge, UK) and BD Pharmingen, respectively. Non-phosphorylated TBK1 peptide was obtained from Sir Philip Cohen (University of Dundee). LPS from Escherichia coli, serotype EH100, was from Alexis (San Diego, CA), poly(I-C) was from Amersham Biosciences, and dexamethasone (D4902) was purchased from Sigma. RANTES enzyme-linked immunosorbent assay kit was from R&D Systems (Abingdon, UK). Cell Culture and Transient Transfection—Human peripheral blood mononuclear cells (PBMC) were isolated from human blood and maintained in RPMI supplemented with 10% fetal calf serum, 2 mm l-glutamine, 1% penicillin/streptomycin solution (v/v). U373 astrocytoma cells stably transfected with CD14 (U373-CD14) were a kind gift from Katherine Fitzgerald (University of Massachusetts Medical School) and were grown in Dulbecco's modified Eagle's medium supplemented as above with the addition of 250 μg/ml neomycin analog G418 to maintain CD14 expression. For transfections, U373-CD14 cells were seeded in 24-well plates at 3 × 104 cells per well (for ISRE luciferase assays) or in 6-well plates at 1.2 × 105 per well (for Western blot analysis), incubated overnight, and transfected using GeneJuice transfection reagent (Novagen, Madison, WI) according to the manufacturer's instructions. For ISRE luciferase assays, 75 ng of ISRE luciferase plasmid, 30 ng of Renilla luciferase, and empty pcDNA3.1 vector made up to a total of 220 ng of DNA were transfected into each well of a 24-well plate. For Western blot analysis varying amounts of pCMV-MKP1 plasmid (100 ng, 1 μg, or 2 μg) and empty pcDNA3.1 vector made up to a total of 2 μg of DNA was transfected into each well of a 6-well plate. In both cases cells were transfected for 24 h before treatment with dexamethasone and stimulation with LPS (100 ng/ml) or poly(I-C) (50 μg/ml) as indicated in the figure legends. ISRE Luciferase Assays—Cells were lysed in 100 μl of passive lysis buffer (Promega, Southampton, UK) for 15 min. Firefly luciferase activity was assayed by the addition of 40 μl of luciferase assay mix (20 mm Tricine, 1.07 mm (MgCO3)4Mg(OH)2·5H2O, 2.67 MgSO4, 0.1 m EDTA, 33.3 mm dithiothreitol, 270 mm coenzyme A, 470 mm luciferin, 530 mm ATP) to 20 μl of the lysed sample. Renilla luciferase was read by the addition of 40 μl of a 1:1000 dilution of Coelentrazine (Argus Fine Chemicals) in phosphate-buffered saline. Luminescence was read using the Reporter microplate luminometer (Turner Designs). The Renilla luciferase plasmid was used to normalize for transfection efficiency in all experiments. Western Blot Analysis—Human PBMC and U373-CD14 cells were seeded in 6-well plates at 3 × 106 and 1.2 × 105 per well, respectively. Cells were treated with dexamethasone and stimulated with LPS (100 ng/ml) or poly(I-C) (50 μg/ml) as indicated in the figure legends. Cells were washed in ice-cold phosphate-buffered saline before being lysed on ice in 100 μl of low stringency lysis buffer (50 mm Hepes, pH 7.5, 100 mm NaCl, 10% glycerol (v/v), 0.5% Nonidet P-40 (v/v), 1 mm EDTA containing 1 mm dithiothreitol, 1 mm sodium orthovanadate, 50 mm sodium fluoride, 5 mm sodium pyrophosphate, 0.1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, and 1 μg/ml leupeptin). The cell lysates were centrifuged at 13,000 rpm for 15 min after which the supernatants were removed and determined for protein concentration using Coomassie Bradford reagent according to manufacturer's instructions (Pierce). Samples containing equal protein concentrations were generated using 5× SDS sample loading buffer (125 mm Tris-HCl, pH 6.8, 15% glycerol (v/v), 2% SDS (v/v), 10 mg bromphenol blue) containing 50 mm dithiothreitol. SDS protein samples (25 μg) were resolved on 8 or 10% SDS-PAGE gels and transferred onto nitrocellulose membranes. Membranes were blocked in 5% (w/v) dried milk in TBS-T (50 mm Tris/HCl, pH 7.6, 150 mm NaCl, and 0.1% (v/v) Tween 20), and incubation of primary antibody was carried out in the same buffer with the exception of phospho-Ser-396 IRF3 and phospho-p38 MAP kinase, which were incubated in 2.5% bovine serum albumin (w/v)/TBS-T. Blots were then incubated with the appropriate secondary antibody in 5% (w/v) dried milk/TBS-T before being developed by enhanced chemiluminescence (ECL) according to the manufacturer's instructions (Cell Signaling Technology, Inc.). Densitometric analysis of band intensities was determined using Multi Gauge Version 2.2 software. Native PAGE for Analysis of IRF3 Dimerization—Preparation of cell lysates and native gel analysis was performed as previously described (5Iwamura T. Yoneyama M. Yamaguchi K. Suhara W. Mori W. Shiota K. Okabe Y. Namiki H. Fujita T. Genes Cells. 2001; 6: 375-388Crossref PubMed Scopus (236) Google Scholar). Immunoprecipitation Assays—U373-CD14 cells were seeded at 8 × 105 per 10-cm plate. Once confluent, cells were treated as indicated in the figure legends, washed in ice-cold phosphate-buffered saline, lysed in 300 μl of low stringency lysis buffer, and determined for protein concentration as described above. For immunoprecipitation kinase assays, 500–800 μg of total protein was incubated with 2 μg of anti-NAK (TBK1) overnight at 4 °C. Additional assay controls such as FLAG-tagged kinase dead TBK1 (KD), which had been transfected into U373-CD14 cells 24 h before stimulation, was immunoprecipitated with 2 μg of anti-FLAG (Sigma). 2 μg of anti-rabbit IgG was used as an internal immunoprecipitation control. In each case, 10 μl of protein A/G-agarose beads (Santa Cruz Biotechnology) were added for 2 h at 4°C. Beads were washed twice in low stringency lysis buffer followed by one wash in kinase buffer (20 mm Hepes, pH7, 10 mm MgCl2, 50 mm NaCl containing 1 mm dithiothreitol, 1 mm sodium fluoride, 0.1 mm sodium orthovanadate, and 0.1 mm phenylmethylsulfonyl fluoride). The beads were then incubated for 25 min at 30 °C in a kinase reaction with 1 μg of recombinant substrate GST-IRF3 (380–427), GST-IRF3 (5A) or GST-IRF3 (7A), 1 mm ATP and 5 μCi [γ-32P]ATP (Amersham Biosciences) made up to a total volume of 20 μl with kinase buffer. Samples were then resolved by SDS-PAGE. The upper half of the gel was transferred onto polyvinylidene difluoride and blotted for total TBK1 (Imgenex), whereas the bottom half was stained with Coomassie to detect substrate GST-IRF3 and subsequently exposed to an (X-ray film) overnight at –80 °C. For Ser(P)-172-TBK1 assays, 500–800 μg of total protein was incubated with 2 μg of anti-TBK1 (Ser-172) and 2 μg of nonphosphorylated TBK1 peptide overnight at 4 °C after which 10 μl of protein A/G-agarose beads were added for 2 h at 4 °C. The beads were washed 3 times in low stringency lysis buffer and resuspended in 20 μl of 2× SDS sample loading buffer and analyzed for total TBK1 by Western blot analysis. Dexamethasone Inhibits the Induction of RANTES and an ISRE Reporter Gene by TLR4 and TLR3—We first investigated if dexamethasone could affect the functional outcomes of endogenous IRF3 activity in response to both TLR4 and TLR3 stimulation. We, therefore, analyzed the effects of dexamethasone on RANTES expression in response to LPS and poly(I-C) since phosphorylation of IRF3 is essential for RANTES induction (18McWhirter S.M. Fitzgerald K.A. Rosains J. Rowe D.C. Golenbock D.T. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 233-238Crossref PubMed Scopus (450) Google Scholar). As shown in Fig. 1A, stimulation of U373-CD14 cells with LPS (left-hand panel) or poly(I-C) (right-hand panel) induced a 7–8-fold increase in RANTES expression from a basal 100 pg/ml to 700–800 pg/ml. Dexamethasone dose-dependently inhibited this response, with 10 μm decreasing RANTES induction by 90%. We also investigated whether dexamethasone had the same effect on RANTES expression in primary cells. Human PBMC produced a 7-fold increase in RANTES expression in response to LPS (Fig. 1B, left-hand panel) and a 2–3-fold increase in response to poly(I-C) (right-hand panel). In both cases dexamethasone markedly inhibited this response. We next analyzed the effect of dexamethasone on an ISRE luciferase reporter plasmid, which contains five repeats of the ISRE sequence. Stimulation of cells with LPS and poly(I-C) resulted in a 6- and 4-fold increase of ISRE luciferase activity, respectively (Fig. 1C). Dexamethasone dose-dependently inhibited both stimuli with an optimal effect evident at 10 μm. These results indicate that the effect of dexamethasone on IRF3-dependent genes is unlikely to be specific to LPS. Dexamethasone Inhibits IRF3 Phosphorylation in Response to LPS and Poly(I-C)—We next examined whether dexamethasone had any effect on the phosphorylation status of IRF3. Phosphorylated IRF3 exists in multiple forms that have been previously characterized (4Servant M.J. ten Oever B. LePage C. Conti L. Gessani S. Julkunen I. Lin R. Hiscott J. J. Biol. Chem. 2001; 276: 355-363Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). In a resting cell IRF3 can be visualized as a doublet on SDS-PAGE (Fig. 2A, upper panels) where the lower and upper band have been named form I and form II, respectively. Form I represents non-phosphorylated IRF3, whereas form II represents a basally phosphorylated IRF3 (Fig. 2A, left side, upper panel, lane 1). Here we show that treatment of U373-CD14 cells with LPS over time induces a band shift to form II, which is maximal at 60 min (Fig. 2A, left side, upper panel, lane 7). This band shift to form II is indicative of IRF3 N-terminal phosphorylation (4Servant M.J. ten Oever B. LePage C. Conti L. Gessani S. Julkunen I. Lin R. Hiscott J. J. Biol. Chem. 2001; 276: 355-363Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 18McWhirter S.M. Fitzgerald K.A. Rosains J. Rowe D.C. Golenbock D.T. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 233-238Crossref PubMed Scopus (450) Google Scholar). Pretreating the cells with dexamethasone inhibited this response (compare lane 6 to lane 5 for 30 min LPS and lane 8 to lane 7 for 60 min of LPS). To establish if LPS could cause phosphorylation of any of the C-terminal residues, the same lysates were immunoblotted for Ser-396, a critical residue for IRF3 activation (9Servant M.J. Grandvaux N. tenOever B.R. Duguay D. Lin R. Hiscott J. J. Biol. Chem. 2003; 278: 9441-9447Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 14Clement J.F. Bibeau-Poirier A. Gravel S.P. Grandvaux N. Bonneil E. Thibault P. Meloche S. Servant M.J. J. Virol. 2008; 82: 3984-3996Crossref PubMed Scopus (71) Google Scholar). Phosphorylation of IRF3 on Ser-396 occurred from 30 min after LPS stimulation and was maximal at 60 min, which correlated with the band shift observed with the total IRF3 antibody (Fig. 2A, left side, second panel, lanes 5 and 7). Pretreating the cells with 1 μm dexamethasone again inhibited this response (compare lane 6 to lane 5 for 30 min of LPS and lane 8 to lane 7 for 60 min of LPS). We next examined the effect of dexamethasone on poly(I-C)-induced IRF3 phosphorylation. Despite repeated attempts we were unable to detect a band shift in IRF3 on SDS-PAGE analysis (Fig. 2A, right side, upper panel), which may be explained by the fact that poly(I-C) has never been found to induce N-terminal phosphorylation of IRF3. However, we were able to detect increased phosphorylation of Ser-396 after a treatment time of 90 min (Fig. 2A, right side, second panel, lane 9). Importantly this was inhibited with dexamethasone pretreatment (lane 10). As a positive control for dexamethasone, lysates were also immunoblotted for phospho-p38, which has previously shown to be inhibited by dexamethasone (25Lasa M. Abraham S.M. Boucheron C. Saklatvala J. Clark A.R. Mol. Cell. Biol. 2002; 22: 7802-7811Crossref PubMed Scopus (319) Google Scholar). Both LPS and poly(I-C) strongly induced p38 phosphorylation where the LPS effect was evident at 15 min (Fig. 2A, left side, third panel, lane 3), and poly(I-C) caused an effect from 60 min (Fig. 2A, right side, third panel, lane 7). As expected, dexamethasone could inhibit p38 phosphorylation in response to both stimuli at all time points (e.g. compare lane 4 to lane 3 for 15 min LPS, and compare lane 8 to lane 7 for 60 min poly(I-C)). The effects of dexamethasone were further analyzed by performing both a time course and a dose response (Fig. 2, B and C). In the time course, cells were incubated with dexamethasone for either 2, 6, or 24 h before being stimulated with LPS for 60 min (Fig. 2B, left side) or poly(I-C) for 90 min (Fig. 2B, right side). At all incubation time points, dexamethasone could reduce the ability of LPS to cause a band shift in IRF3 to form II (Fig. 2B, left side, upper panel), and this appeared to be maximal when the cells were pretreated with dexamethasone for 24 h (lane 8). Optimal inhibition of LPS-induced phosphorylation of Ser-396 was also maximal at a pretreatment time of 24 h (Fig. 2B, left side, second panel, lane 8). As mentioned previously, poly(I-C) failed to induce an IRF3 band shift; however, activation could be monitored by immunoblotting for Ser-396. Dexamethasone dramatically reduced Ser-396 phosphorylation at each time point, with pretreatment for 24 h causing the greatest effect (Fig. 2B, right side, second panel, lane 8). For the dose response, cells were treated with increasing doses of dexamethasone (1 nm to 10 μm) for 24 h before stimulation with LPS for 60 min or poly(I-C) for 90 min (Fig. 2C). This resulted in a dose-dependent reduc

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