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J. Biol. Chem., Vol. 282, Issue 20, 15022-15032, May 18, 2007
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Is Involved in Interferon Regulatory Factor 3 Activation and Type I Interferon-
Synthesis*
From the Institute for Medical Immunology, Université Libre de Bruxelles (U. L. B.), 8 Rue Adrienne Bolland, 6041 Charleroi, Belgium
Received for publication, January 16, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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in the regulation of IFN-
gene expression mediated by the Toll-like receptor 3 (TLR3) signaling pathway. Inhibition of conventional PKC (cPKC) activity in monocyte-derived dendritic cells or TLR3-expressing cells by an isoform-specific inhibitor, Gö6976, selectively inhibited IFN- synthesis induced by double-stranded RNA polyinosine-polycytidylic acid. Furthermore, reporter gene assays confirmed that PKC regulates IFN- promoter activity, since overexpression of dominant negative PKC but not PKCI repressed interferon regulatory factor 3 (IRF-3)-dependent but not NF-
B-mediated promoter activity upon TLR3 engagement in HEK 293 cells. Dominant negative PKC inhibited IRF-3 transcriptional activity mediated by overexpression of TIR domain-containing adapter inducing IFN- and Tank-binding kinase-1. Additional biochemical analysis demonstrated that Gö6976-treated dendritic cells exhibited IRF-3 phosphorylation, dimerization, nuclear translocation, and DNA binding activity analogous to their control counterparts in response to polyinosine-polycytidylic acid. In contrast, co-immunoprecipitation experiments revealed that TLR3-induced cPKC activity is essential for mediating the interaction of IRF-3 but not p65/RelA with the co-activator CREB-binding protein. Furthermore, PKC knock-down with specific small interfering RNA inhibited IFN- expression and down-regulated IRF-3-dependent promoter activity, establishing PKC as a component of TLR3 signaling that regulates IFN- gene expression by targeting IRF-3-CREB-binding protein interaction. Finally, we analyzed the involvement of cPKCs in other signaling pathways leading to IFN- synthesis. These experiments revealed that cPKCs play a role in the synthesis of IFN- induced via both TLR-dependent and -independent pathways. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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and the IFN- family are central to the innate immunity of mammals and the development of effective adaptive immune responses against viruses and tumors (1, 2). Toll-like receptors (TLRs) expressed by innate immune cells, including dendritic cells (DCs), recognize distinct pathogen-associated molecular patterns, leading to the activation of innate immunity, which shapes the subsequent adaptive immune response (3, 4). TLR3 triggering by double-stranded RNA generated during viral infection with synthetic double-stranded RNA analog poly(I:C), endogenous cellular mRNA structures, and small interfering RNAs results in DC activation to promote type I IFNs (5-7). Recent studies have demonstrated the involvement of TLR3 in antiviral defense and its prominent role in the cross-priming mechanism, since DCs from TLR3-deficient mice exhibit reduced inflammatory responses mediated by reovirus genomic double-stranded RNA and poly(I:C) and are compromised in CTL cross-priming (5, 8-10).
The signaling pathway downstream of TLR3 requires the adaptor molecule TRIF that recruits tumor necrosis factor-associated factor-6 to channel NF-B activation and mediate the phosphorylation of the two noncanonical IB kinases, IB kinase-
and Tank-binding kinase 1 (TBK1) (9, 11-13). Uncoupling at the level of TRIF, TBK1, and IKK kinases induces events leading to IRF-3 phosphorylation-dependent activation (12, 14). Importantly, both of these phosphorylate distinct C-terminal Ser residues on IRF-3, which results in IRF-3 homodimerization, nuclear translocation, and enhanced ability to bind to the IFN- promoter (6, 15). Upon activation, IRF-3 homodimers associate with the transcriptional co-activators CREB-binding protein (CBP) and p300 in the nucleus, an event that enables the optimal induction of the IFN- promoter (16-19).
The enhancer region of the IFN- promoter has been extensively characterized as containing four overlapping regulatory elements that are designated as positive regulatory domain (PRD)-I, -II, -III, and -IV (17, 19, 20). Promoter region analysis of the Ifn gene revealed that PRDII and PRDIV are bound by NF-B and ATF-2/c-Jun, respectively, and that these elements cooperate with PRDI and PRDIII in the induction of the IFN- promoter (21). The PRDI and PRDIII elements were shown to be bound by distinct members of the IRF family with much attention focused on IRF-3, which binds to the PRDIII-I composite site on the IFN- promoter and is required for IFN- gene transcription (22-24).
PKCs mediate an evolutionarily conserved function in host defense against fungal and bacterial infections from primitive organisms up to mammals (25, 26). The PKC family subdivides into three main groups based on the presence or absence of distinct motifs determining co-factor requirements for their activity. Conventional PKCs (, I, II, and 
) require Ca2+ binding and are activated by diacylglycerol and phorbol esters, whereas novel PKCs (
, , and 
) do not require Ca2+ but are also diacylglycerol/phorbol ester-sensitive. The last subgroup, atypical PKC (
and 
/
) require neither Ca2+ nor diacylglycerol/phorbol esters (27, 28). Previous studies demonstrated that PKC isoforms regulate several signaling pathways, including innate immune responses induced by microbial products (26). Indeed, Gram-negative LPS was shown to activate distinct PKC isoforms in DCs and macrophages (M
s) (29-33). Specifically, PKC is an essential integrator of LPS-mediated inflammatory cytokine production through activation of NF-B and mitogen-activated protein kinases (29, 34). Importantly, PKC-deficient Ms display defects in clearing both Gram-negative and positive bacterial infections (34). Although PKC involvement in poly(I:C)-mediated innate responses was previously observed using broad range pharmacological inhibitors (35, 36), participation of PKC isoforms in poly(I:C)-mediated activation of TLR3 pathway still remains unknown.
In this work, we investigated the role of cPKC isoforms in poly(I:C)-induced cytokines, particularly IFN- and the molecular targets downstream of the TLR3 signaling pathway. Cellular inhibition assays, using a conventional PKC inhibitor, Gö6976, performed in monocyte-derived DCs, HEK 293, and HEK 293 cells stably expressing TLR3 demonstrated that conventional PKC activity is involved in IFN- gene expression. In support of this observation, we showed that overexpression of dominant negative (DN) PKC as well as Gö6976 inhibits IRF-3-dependent but not NF-B-mediated reporter gene activities, correlating with repression of IFN- promoter activity. Further biochemical analysis revealed an unexpected role for cPKC activity that is essential for mediating IRF-3 association with CBP. Particularly, PKC knockdown by siRNA in TLR3-293 cells established that PKC participates in IRF-3-mediated IFN- gene expression. Finally, our data show that cPKCs play a general role in IFN- synthesis induced either by TLR-dependent or TLR-independent pathways.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Stable Cell Lines and Reagents—Human embryonic kidney (HEK) 293 parental cells and cells stably expressing FLAG-TLR3 (TLR3-293) were kindly provided by S. Akira (Osaka University, Osaka, Japan). The multimerized NF-B was kindly provided by W. Vanden Berghe (University of Gent); PRDIII-I (12), IFN- promoter (IFN- pGL-3) (39), Gal4-luciferase reporter construct, and Gal4-IRF-3 expression vector were described earlier (16). E-tag-TRIF was a gift from R. Beyaert (University of Gent). pHACE-PKC-KR (DN) expression plasmids were generated by ligating full-length open reading frames of PKC isoforms with a K368R point mutation at the ATP binding site into pHACE digested with EcoRI (40). Poly(I:C) was purchased from Amersham Biosciences, ds--DNA was purchased from InvivoGen (Toulouse, France), and Gö6976 was from Biomol (Boechout, Belgium). Granulocytemacrophage colony-stimulating factor was obtained from LEUCOMAX (Schering-Plough, France), and IL-4 was from R&D Systems (Abingdon, United Kingdom).
Transfection Assays—HEK 293 or TLR3-293 cells were seeded in 24-well plates at a density of 5 x 105/ml. The following day, cells were transfected with 1 µg of the indicated luciferase reporter plasmids, using FuGENETM-6 (Roche Applied Science) according to the manufacturer's specifications. All transfections included 40 ng of Renilla luciferase DNA in the pRL-TK vector (Promega, Leiden, The Netherlands) as an internal control. Where indicated, the cells were stimulated with poly(I:C) (50 µg/ml) for 18 h, and subsequently the cells were harvested and the whole cell extracts (WCEs) were obtained, followed by analysis of promoter activities using the dual luciferase reporter assay system (Promega). Promoter activities were normalized to Renilla luciferase activities. Data are expressed as the mean relative stimulation ± S.D.
siRNA Transfection—Duplexed siRNAs targeting human PKC and GFP, each labeled with 3' Alexafluor 647, were purchased from Qiagen (KJ Venlo, The Netherlands). HEK 293 cells stably expressing TLR3 were seeded in 24-well plates at a density of 1.25 x 105. Twenty-four hours later, cells were transfected with the indicated concentrations of PKC siRNA or control, GFP siRNA using X-treme gene (Roche Applied Science) according to the manufacturer's protocol. After 48 h of incubation, the supernatant was removed and replaced with fresh medium and subsequently stimulated with poly(I:C) (10 µg/ml). The cells were collected after 18 h of stimulation, and the efficiency of PKC knockdown was assessed by Western blotting. The cell supernatants were analyzed for IFN- and IL-8 production, as described below. For DNA/RNA co-transfection studies, TLR3-293 cells (1.25 x 105/ml) were transfected with 1 µg of IRF-3-Gal4 reporter plasmid using X-treme gene (Roche Applied Science). 24 h later, PKC- or GFP-siRNA was added. After 48 h of incubation, the supernatant was removed and replaced with fresh medium. Where indicated, the cells were stimulated with poly(I:C) (10 µg/ml) for 18 h, and WCEs were harvested. Promoter activities were analyzed as described above.
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-DNA, using FuGENETM-6 (Roche Applied Science) according to the manufacturer's specification, for 20 h. IFN- levels were measured with ELISA kits from BIOSOURCE (Nivelles, Belgium). Quantification of IL-8 and tumor necrosis factor- were performed using antibodies from BIOSOURCE.
RNA Purification and Quantitative Reverse Transcription-PCR—Total cellular mRNA from DC (1 x 106/ml) was extracted using a MagnaPure LC mRNA isolation kit (Roche Applied Science) according to the manufacturer's specification. Reverse transcription and quantitative reverse transcription-PCR were performed using Light-Cycler-RNA Master Hybridization probes (one-step procedure) on a Lightcycler® apparatus (Roche Applied Science). The conditions were previously described (38). The oligonucleotide sequences used for amplification of the IFN- were as follows: sense primer, 5'-GGATGCAGGAAGGAGATCACTG-3'; anti-sense primer, 5'-CGATCCACACGGAGTACTTG-3'; probe, 5'-(6-carboxyfluorescein succinimidyl ester)CCCTGGCACCAGCACAATG(6-carboxytetramethylrhodamine succinimidyl ester) (phosphate)-3'. The oligonucleotides used to amplify -actin were described earlier (38).
Immunoblotting—DC or TLR3-293 cells (1 x 105/ml) were collected and directly lysed in Laemmli buffer. Equal volumes of WCEs from each condition were resolved by 8% SDS-PAGE and analyzed by Western blotting. Immunoblots were probed with polyclonal antibodies directed against phosphopan-PKC (Cell Signaling, Leusden, the Netherlands), phospho-IRF-3 Ser396, kindly provided by R. Lin (Lady Davis Institute for Medical Research, McGill University), PKC (Santa Cruz Biotechnology, Inc.), PKCI (Santa Cruz Biotechnology), anti-HA (Euroscreen, Rixensart, Belgium), phospho-ATF-2 (Cell Signaling), or phospho-c-Jun (Cell Signaling). Equal loading was verified either by anti-PKC (Santa Cruz Biotechnology), anti-IRF-3 (BD Biosciences), an anti-p65 (SC-372, Santa Cruz Biotechnology), ATF-2 (Santa Cruz Biotechnology), c-Jun (Santa Cruz Biotechnology), or glyceraldehyde-3-phosphate dehydrogenase antibodies (Biodesign International, Saco, ME). The immunoreactive bands were revealed using the ECL® detection kit (Amersham Biosciences). For co-immunoprecipitation studies, WCEs (400 µl) of DCs or TLR3-293 cells were lysed in buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, pH 8.0, 30 mM NaF, 1 mM Na3Vo4, 40 mM -glycerophosphate, 0.1 mM phenylmethylsulfonyl fluoride in EtOH, protease inhibitor mixture (Roche Applied Science), 10% glycerol, and 1% Nonidet-P40 (Nonidet P-40)) and then incubated overnight with 1 µg of anti-CBP antibody (C-1; Santa Cruz Biotechnology). WCEs were incubated with 30 µl of a 1:1 slurry of protein A/protein G-Sepharose beads (Amersham Biosciences) for 2 h at 4 °C. The beads were washed five times with lysis buffer and resuspended in 1x Laemmli buffer. The immunoprecipitated complexes and WCEs were analyzed by immunoblotting with an anti-IRF-3 antibody (BD Biosciences) or an anti-p65 antibody (SC-372; Santa Cruz Biotechnology).
Native PAGE—DCs (1 x 106/ml) were collected and lysed in native lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, pH 8.0, 1 mM Na3Vo4, 1 mM -glycerophosphate, 1 mM phenylmethylsulfonyl fluoride in EtOH, protease inhibitor mixture, and 1% Nonidet P-40). The SDS-free 7.5% polyacrylamide gel was prerun with 25 mM Tris, 192 mM glycine with and without 1% deoxycholate in the cathode and anode chamber, respectively, for 30 min at 40 mA. Samples (6 µg) were combined 1:1 with native sample buffer (Bio-Rad), loaded, and electrophoresed for 60 min at 35 mA. Proteins were transferred to membranes and probed for IRF-3 dimerization with monoclonal antibody IRF-3 (BD Biosciences).
Determination of NF-B and IRF-3 DNA Binding Activities— Nuclear extracts were prepared as described earlier (41). IRF-3 or NF-B DNA-binding activities in nuclear extracts were measured by Trans-AM IRF-3 or p65 transcription factor assay kits (Active Motif Europe, Rixensart, Belgium) according to the manufacturer's protocols. Briefly, 3 µg of each nuclear extract was incubated in plates coated with consensus IFN-stimulated response elements and NF-B oligonucleotides, respectively. Plates were washed, and anti-IRF-3 or p65 antibodies were added to the wells. Antibody binding was detected with a secondary horseradish peroxidase-conjugated antibody and developed with TMB substrate. The intensity of the reactions was measured at 450 nm.
Confocal Microscopy—DCs (2 x 105) were seeded on glass coverslips and allowed to adhere for 1 h. Following cellular activation, DCs were fixed in 2% paraformaldehyde and permeabilized with 100% cold methanol and then were incubated in blocking buffer (phosphate-buffered saline, 2% bovine serum albumin) for 20 min. The cells were incubated with IRF-3 monoclonal antibody (5 µg/ml) (BD Biosciences) followed by p65/RelA (4 µg/ml) (Santa Cruz Biotechnology) and detected with ALEXA-488- and ALEXA-568-conjugated secondary antibodies (2 µg/ml) from Invitrogen. Following RNase treatment, nuclei were stained with TOTO®-3 iodide (5 µM) (Invitrogen). The cells were mounted in Vectashield mounting medium (Vector Laboratories Inc., Brussels, Belgium) and visualized with a Leica TCS SP2 AOBS confocal microscope (Leica, Deerfield, IL). Images were acquired using immersion oil objective x100 HCV PL APO CS with a x1.40 numerical aperture. The scanned images were acquired with Leica confocal software.
Statistical Analysis—Data were compared using paired Wilcoxon's nonparametric test or Student's t test (where indicated).
Supplemental Material—Fig. S1 shows that inhibition of conventional PKC activity suppresses IFN- production mediated by the engagement of TLR3.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Synthesis by a Conventional PKC Inhibitor—Engagement of distinct TLRs by their specific ligands induces activation of several PKC isoforms in various cell types (26). Therefore, we first assessed whether poly(I:C) induced the activation of PKC isoforms in monocyte-derived DCs. As shown in Fig. 1A, DCs exhibited phosphorylated forms of conventional PKC/ and novel PKC isoforms within 30 min following the poly(I:C) encounter. Gö6976, a potent and selective conventional PKC inhibitor targeting Ca2+-dependent PKC isoforms (42), completely abolished the phosphorylation-dependent activation of conventional PKC isoforms without effecting PKC activation.
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secretion from DCs. Next, we analyzed whether the inhibitory effect of Gö6976, observed on IFN- protein secretion, was due to an effect on IFN- mRNA transcription. Using quantitative reverse transcription-PCR analysis, we observed that the accumulation of IFN- mRNA transcripts occurred as early as 2 h, peaking maximally at 4 h following poly(I:C) stimulation (data not shown). At 4 h, Gö6976-treated poly(I:C)-activated DCs exhibited significantly reduced IFN- mRNA levels in comparison with poly(I:C)-activated control counterparts (Fig. 1C). In contrast, poly(I:C) exposure of DCs resulted in IL-8 and tumor necrosis factor- production that was comparable between the Gö6976-treated cells and their control counterparts (Fig. 1, D and E).
PKC Is Involved in TLR3-mediated Induction of IRF-3-dependent Promoter Activities—In the next set of experiments, we investigated the effects of inhibiting poly(I:C)-induced conventional PKC activity on human IFN- promoter activity in TLR3-expressing 293 cells. As shown in Fig. 2, poly(I:C) stimulation of TLR3-293 cells resulted in a strong induction of IFN- promoter activity, which was repressed 3-fold in Gö6976-treated counterparts.
Our data pointed out that cPKCs were involved in IFN- gene expression through regulation of IFN- promoter activity. Furthermore, in two distinct cell types, we found that cPKC signaling is selectively involved in the regulation of IFN- but not inflammatory cytokines upon poly(I:C) encounter. Since IFN- gene transcription requires the cooperation of NF-B (12, 43, 44) and IRF-3 transcription factors (22-24), we investigated the involvement of conventional PKC isoforms in the regulation of IRF-3- and/or NF-B-mediated transcriptional activities using either IRF-3- or NF-B-dependent promoters in TLR3-293 cells, which also express PKC and PKCI conventional isoforms endogenously (Fig. 3A). We transfected TLR3-293 cells with either Gal4-IRF-3, the PRDIII-I site from the IFN- promoter, or a multimerized NF-B site from the IL-8 promoter in the presence of increasing concentrations of Gö6976 and subsequently stimulated it with poly(I:C). In Fig. 3B (top and middle), we demonstrated that the Gö6976 treatment resulted in a dose-dependent repression of poly(I:C)-mediated Gal-4-IRF-3 reporter activity as well as transcription mediated at the PRDIII-I site in TLR3-293 cells. In order to assess which of the conventional PKC isoforms are involved in TLR3 signaling, we overexpressed DN PKC and PKCI isoforms in TLR3-293 cells. As shown in Fig. 3C (top), IRF-3 reporter activity was dose-dependently inhibited (
6-fold at the highest dose) by DN PKC but not by DN PKCI overexpression. In a similar setting, poly(I:C)-induced reporter gene transcription at the PRDIII-I site from the IFN- promoter was similarly inhibited (Fig. 3C, middle). Strikingly, poly-(I:C)-induced NF-B reporter gene activity from the IL-8 promoter was altered by neither DN PKC nor PKCI expression vector nor Gö6976 treatment (Fig. 3, B and C, bottom). In the reporter assay settings we tested, DN PKCI had no effect while being optimally expressed in HEK 293 cells (Fig. 3A, right). Hence, we concluded that PKC selectively regulates transcriptional activity of IRF-3-dependent promoters induced by poly(I:C) triggering of TLR3.
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Is Involved in TRIF-mediated IRF-3 Activation Downstream of TBK1—Transcriptional activity at the PRDIII-I site from the IFN- promoter requires TRIF interaction with TBK1, which channels phosphorylation-dependent activation of IRF-3 (12, 45, 46). Next we investigated whether TRIF-mediated and TBK1-mediated signaling to IRF-3 are targeted by conventional PKC isoforms. To assess this question, we analyzed the effects of DN PKC and DN PKCI isoforms on IRF-3-mediated reporter gene activity induced by TRIF or TBK1 overexpression in HEK 293 parental cells. As shown in Fig. 4A (top), TRIF overexpression resulted in the increase of IRF-3-dependent reporter gene activity. Co-expression of DN PKC but not DN PKCI dose-dependently inhibited (3-fold at the highest dose) TRIF-mediated IRF-3 transcriptional activity. In Fig. 4A (bottom), TRIF-mediated induction of reporter gene activity at the PRDIII-I site in 293 cells was inhibited (6-fold at the highest dose) by DN PKC, whereas DN PKCI had no effect. Furthermore, DN PKC (but not DN PKCI) overexpression dose-dependently repressed IRF-3-mediated reporter gene activity as well as transcription at the PRDIII-I site mediated by TBK1 (Fig. 4B, top and bottom). Taken together, these results indicated that PKC participates in the optimal induction of IRF-3 transcriptional activity downstream of TRIF and its immediate effector kinase TBK1.
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gene expression and IRF-3-dependent transcriptional activity led us to investigate the functional outcomes of inhibiting conventional PKCs on early signaling events important for IRF-3 activation. Poly(I:C)-mediated site-specific IRF-3 Ser396 phosphorylation is critical for IRF-3 nuclear translocation and DNA binding on IFN-stimulated response element sites on IRF-3-responsive promoters (39, 47). Therefore, we first examined the phosphorylation status of IRF-3 at Ser396 in the presence or absence of Gö6976 in poly(I:C)-activated DCs. Interestingly, Western blotting experiments demonstrated that poly(I:C)-mediated phosphorylation of IRF-3 at Ser396 residue was comparable whether or not DCs were exposed to Gö6976, shown by a slower migrating band (Fig. 5A). Following phosphorylation, IRF-3 forms a homodimer, an important process required for its transcriptional activity (39, 48, 49). We therefore analyzed the effects of inhibiting conventional PKCs on IRF-3 homodimer formation in DC extracts by native PAGE analysis. Our data demonstrated that conventional PKC inhibitor Gö6976 does not influence the formation of IRF-3 homodimers that occur following poly(I:C) encounter by DCs (Fig. 5B).
Next, using confocal microscopy, we showed that inhibition of conventional PKC activity did not affect poly(I:C)-mediated nuclear localization of IRF-3 in DCs, since poly(I:C)-induced IRF-3 nuclear translocation was comparable between Gö6976-treated DCs and the vehicle-treated control counterparts (Fig. 5C, top). As displayed in Fig. 5C (middle), Gö6976-treated DCs exhibited similar levels of poly(I:C)-induced p65/RelA translocation in comparison with their control counterparts. Finally, we assessed poly(I:C)-induced IRF-3 DNA binding activity in DCs exposed to Gö6976. IRF-3 DNA binding was increased in nuclear extracts from DCs, detected at 90 min following poly(I:C) stimulation (Fig. 5D, left). The level of IRF-3 DNA binding in Gö6976-treated DCs was not significantly altered. Likewise, poly(I:C)-induced NF-B DNA binding was also not affected by Gö6976 treatment, indicating that neither NF-B nuclear translocation nor its DNA binding is influenced by Gö6976 (Fig. 5D, right). Hence, our data indicated that the modifications rendered by inhibiting conventional PKC activity in TLR3 signaling do not involve previously documented events leading to IRF-3 activation, specifically dimerization, nuclear translocation, and DNA binding ability.
Inhibition of Conventional PKC Activity Hinders IRF-3 Binding to Co-activator CBP—Thus far, we have shown that PKC participates in TLR3-mediated transcriptional activity of IRF-3-dependent promoters. Given that conventional PKC inhibitor, Gö6976, diminishes IFN- expression without influencing the initial events important for IRF-3 phosphorylation, homodimerization, nuclear translocation, and DNA binding, we investigated whether conventional PKCs are important for IRF-3 association with the co-activator CBP. In co-immunoprecipitation experiments, we observed a robust physical interaction between the endogenous forms of IRF-3 and CBP in poly(I:C)-stimulated DCs that was severely decreased in Gö6976-treated counterparts (Fig. 6A, left). Similar results, showing strongly reduced IRF-3 interaction with CBP, were obtained from TLR3-293 cells exposed to Gö6976 (Fig. 6B, left). Previously, the co-activator CBP was shown to interact with other transcription factors, including the p65/RelA member of NF-B (50). To find out whether the effects of Gö6976 resulting in the hindrance of IRF-3 interaction with CBP prevent CBP from binding to NF-B members, we performed co-immunoprecipitation assays to visualize p65 interaction with CBP. Strikingly, poly(I:C)-mediated interaction between p65 and CBP remained intact whether or not DCs or TLR3-293 cells were exposed to Gö6976 (Fig. 6, A and B, right). Overall, these observations indicated that inhibition of conventional PKC activity abrogates IRF-3 but not NF-B interaction with the co-activator CBP.
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induction. As shown in Fig. 7A, TLR3-293 cells exhibited a phosphorylated form of ATF-2 within 30 min following poly(I:C) exposure, which persisted up to 60 min after stimulation and was not affected by Gö6976 treatment. Similarly, the poly(I:C)-induced phosphorylation of c-Jun occurred in an identical kinetic and was maintained even in the presence of Gö6976 (Fig. 7B). Next, using co-immunoprecipitation experiments, we examined the role of cPKCs on ATF-2- or c-Jun-CBP interactions in the same system. TLR3-293 cells exhibited basal ATF-2 or c-Jun-CBP interaction but we were not able to detect an increase of such interaction after poly(I:C) stimulation. Nevertheless, the basal interaction between ATF-2 or c-Jun and CBP was not disrupted by Gö6976 (Fig. 7C). We then performed additional experiments in human DCs; in this setting, the increase of ATF-2 or c-Jun and CBP interaction was still barely detectable even upon poly(I:C) stimulation. However, in a single experiment, where the induction was clearly apparent, the addition of the drug did not affect the ATF-2- or c-Jun-CBP interaction (data not shown).
PKC Is Essential for IFN- Expression in Response to Poly(I:C)—To further confirm the role of PKC in the regulation of IFN- gene expression, we transiently transfected TLR3-293 cells with siRNA targeting PKC. TLR3-293 cells were transfected with PKC-siRNA ranging from 0 to 200 µM. As shown in Fig. 8A (left), PKC knockdown by its target-specific siRNA occurred in a dose-dependent manner, whereas the control GFP-siRNA had no inhibitory effects on PKC protein expression (Fig. 8A, right). Next we investigated the effects of PKC knockdown on IRF-3-mediated transcriptional activity. We co-transfected TLR3-293 cells with a Gal4-IRF-3 promoter and the indicated concentrations of PKC- or GFP-siRNA followed by poly(I:C) stimulation. The poly(I:C)-induced transcriptional activity of Gal4-IRF-3 was inhibited with increasing concentrations of PKC-siRNA, whereas the control GFP-siRNA had no effect on its activity (Fig. 8B). Then we investigated the functional outcomes of PKC knockdown on IFN- expression. TLR3-293 cells were transfected with either PKC or GFP-siRNA and then stimulated with poly(I:C), followed by analysis of cell supernatants for cytokine production by ELISA. IFN- secretion from poly(I:C)-activated TLR3-293 cells was strongly inhibited by the knockdown of PKC whereas the control siRNA had no effect (Fig. 8C, left). However, poly(I:C)-induced IL-8 production was unaffected by either siRNA (Fig. 8C, right), mirroring data obtained with Gö6976 treatment (supplemental Fig. S1, A and B). These data demonstrated that PKC knockdown selectively down-regulates poly(I:C)-mediated IFN- gene expression but does not affect the production of IL-8.
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-DNA-induced IFN- Synthesis—Our data showing the critical role of conventional PKCs in TLR3-mediated IFN- expression led us to investigate the possible involvement of cPKCs on other signaling pathways leading to IFN- synthesis. Therefore, we first assessed the effect of cPKC inhibition on TLR4-mediated IFN- synthesis in DCs. As shown in Fig. 9A, LPS-induced IFN- secretion is significantly inhibited in Gö6976-treated DCs. We next evaluated the effect of inhibiting cPKCs on IFN- synthesis induced by a synthetic ds--DNA, previously described for its ability to trigger a TLR-independent cytosolic pathogen recognition receptor (51). For that purpose, we used HEK 293 cells, which do not express any TLRs. HEK 293 cells were transfected with ds--DNA in the presence of increasing concentrations of Gö6976. As shown Fig. 9B, ds--DNA-induced IFN- was strongly inhibited in a dose-dependent manner, 5-fold at the highest dose. These results revealed that cPKCs are involved in the synthesis of IFN- induced via both TLR4- and TLR-independent pathways. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, which is involved in inflammatory cytokine expression by modulating NF-B activation in human DCs and murine Ms downstream of TRAM. PKC was shown to regulate TLR4 signaling by phosphorylating TRAM, a key process required for its function (29, 33, 34). Furthermore, poly(I:C)-activated PKCs were shown to be involved in IFN- expression, since PKC inhibitors were shown to effectively block type I IFN production in human, simian, and mouse fibroblasts (35, 36).
In this study, we have shown that conventional PKCs, particularly the PKC isoform, participates in poly(I:C)-mediated IFN- gene expression through modulation of IRF-3-dependent transcription in two distinct cell types, human DCs and HEK 293 cells. Inhibition of conventional PKC activity down-regulated IFN- mRNA transcription and repressed IFN- promoter activation in poly(I:C)-activated DCs and TLR3-293 cells, respectively. Moreover, Gö6976-mediated inhibitory effects on IFN- synthesis cannot be attributed to defects in IFNRI-mediated signaling, because inhibition of conventional PKC activity did not modify recombinant type I IFN-mediated phosphorylation of signal transducer and activator of transcription Tyr701 residue (data not shown).
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overexpression as well as PKC-siRNA knockdown establish that TLR3-induced conventional PKC isoform is involved in the regulation/activation of early signaling events that induce activation of the IFN- promoter. Since the cooperative action of IRF-3 and NF-B transcription factors for poly(I:C)-mediated induction of IFN- promoter activity has been well documented (21, 22, 24, 52), we investigated the involvement of conventional PKC and I isoforms in IRF-3- and NF-B-dependent reporter gene transcription. Our data confirm that DN PKC (but not DN PKCI)) effectively repressed IRF-3-dependent but not NF-B-mediated reporter gene activity. Therefore, PKC mediates IRF-3 transcriptional activity but not NF-B-dependent transcription downstream of the TLR3 signaling pathway. In addition, the results from reporter gene assays performed using HEK 293 parental cells demonstrate that DN PKC down-regulates IRF-3 transcriptional activity induced by TRIF or TBK1 overexpression. These findings suggest that PKC operates downstream of TLR3-TRIF-TBK1 and participates in IRF-3 transcriptional activity.
The signaling events controlling poly(I:C)-mediated IRF-3 transcriptional activity are complex and multifaceted. Shortly after poly(I:C) or virus exposure, IRF-3 undergoes a conformational change following phosphorylation of specific serine residues in the C-terminal serinerich region of IRF-3 that allows its homodimerization (53). Following homodimer formation, IRF-3 translocates to the nucleus, where it binds to the PRDIII-I site on IFN- promoter as well as IFN-stimulated response element sites found on IFN-responsive genes (24, 52). The minimal phosphoacceptor site at Ser396 on IRF-3 was shown to be targeted by poly(I:C) signaling, important for IRF-3 translocation and DNA binding ability upon poly(I:C) or viral activation (39). In addition, Fujita and co-workers (16) have demonstrated that point mutations of Ala of Ser385 or Ser386 (referred to as the 2S site), which is part of the serine-rich region, abolishes the phosphorylation and dimerization of IRF-3 by TBK1. Through biochemical analysis, we have assessed the contribution of conventional PKCs in the early signaling events critical for IRF-3 activation, such as (a) IRF-3 phosphorylation, (b) homodimerization, (c) nuclear translocation, (d) DNA-binding ability, and (e) IRF-3 association with CBP/p300. Our data from Western blotting experiments show that poly(I:C)-activated DCs in the presence of Gö6976 exhibited comparable levels of IRF-3 Ser396 and also Ser385/386 phosphorylation (data not shown). In accordance with the lack of effect of conventional PKC inhibition on these critical phospho-Ser residues, native PAGE experiments and confocal microscopy analysis demonstrated that conventional PKCs are not involved in IRF-3 phosphorylation, homodimerization, and nuclear translocation. Our findings concur with previous findings demonstrating that Gö6976 does not affect IRF-3 dimerization or NF-B DNA-binding in poly(I:C)-stimulated HeLa cells (48) or inhibit IRF-3 phosphorylation in response to virus (39, 47).
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B, and ATF-2/c-Jun revealed that CBP/p300 is recruited to the IFN- enhanceosome through distinct interactions with these transcription factors that mediate the maximal induction of transcription at the IFN- promoter (54, 55). It is well established that IRF-3 interaction with CBP occurs between the C terminus of IRF-3 and the IFN-binding domain of CBP (23, 54). Our data from co-immunoprecipitation assays revealed that TLR3-induced conventional PKCs mediate IRF-3 interaction with CBP. Importantly, we showed that inhibition of conventional PKC activity severely hinders the physical interaction between IRF-3 and CBP, which occurs following poly(I:C) exposure. Furthermore, in reporter gene assays, we did not observe any effects of inhibiting conventional PKCs on Gal4-CBP DNA binding activity, indicating that the observed hindrance by Gö6976 of IRF-3-CBP interaction possibly involves an effect on IRF-3 (data not shown). Whether PKC may phosphorylate other unidentified sites involved in IRF-3/CBP interaction or acts indirectly via an unidentified kinase(s) remains to be determined. Overall, our data show evidence that conventional PKCs, particularly PKC, is involved in the interaction of IRF-3 with CBP, which is essential for TLR3-mediated IFN- promoter induction and expression.
Similar to TLR3, TLR4 also mediates type I IFN, using MyD88-independent and TRIF-dependent pathways to induce IRF-3 activation (13, 56). In our system, LPS-induced IFN- production by DCs was likewise sensitive to cPKC inhibition. In conjunction with our data on the TLR3 pathway, this demonstrates a broader role of cPKCs in TLR-dependent signaling. Another important pathway leading to type I IFN synthesis involves the activation of the cytosolic pathway by ds--DNA. Viruses as well as intracellular bacteria that have cytosolic phases in their life cycles are recognized by TLR-independent cytosolic receptors, such as the helicase retinoic acid-inducible gene 1 and melanoma differentiation-associated gene 5, that recognize viral RNAs and synthetic double-stranded RNA (57, 58). These two receptors use the adaptor molecule interferon- promoter stimulator 1 to induce IFN- and cytokines. Subsequently, we investigated the possible role of cPKCs in the induction of IFN- through this TLR-independent pathway. These results revealed that cPKCs are involved in IFN- synthesis induced by both TLR-dependent and TLR-independent pathways.
Although the role of TLR3 in direct priming of antiviral response is controversial, viral evasion of TLR-dependent and -independent pathways argues for the contribution of these signaling pathways in optimal host defense against viruses (9, 59, 60). It is proposed that TLR signaling and the resulting cytokine network plays an active role in the pathogenesis of autoimmunity (2, 61, 62). Type I IFNs, induced by viral infections and/or by ligand-dependent TLR activation, are principal factors that contribute to the breakdown of peripheral tolerance and autoimmune disease generation or progression (2, 62). In this perspective, our study and others (29, 34) demonstrating that distinct PKC isoforms take diverging roles to control TLR signaling pathways highlight the significance of isoform-selective PKC inhibition not only as potential therapy to control inflammation-induced pathologies but also as a target for viral evasion of host immunity. Hence, control of PKC activity may represent a potential strategy to treat autoimmune pathologies originating from TLR-dependent or -independent type I IFN action. Finally, since PKC inhibitors are part of current regimens used for anti-tumor therapy, we suggest that the inhibitory effects of conventional PKC inhibitors on IRF-3 transcriptional activity and IFN- expression should be more cautiously addressed in vivo.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.
1 Supported by the Télévie program of the Fonds National de la Recherche Scientifíque (FNRS).
2 These two authors share senior authorship.
3 To whom correspondence should be addressed. Tel.: 32-2-650-9584; Fax: 32-2-650-9563; E-mail: fwillems{at}ulb.ac.be.
4 The abbreviations used are: IFN, interferon; IRF-3, interferon regulatory factor 3; poly(I:C), polyinosine:polycytidylic acid; siRNA, small interfering RNA; PRD, positive regulatory domain; TLR, Toll-like receptor; WCE, whole cell extract; LPS, lipopolysaccharide; CBP, CREB-binding protein; CREB, cAMP-response element-binding protein; HEK, human embryonic kidney; DN, dominant negative; NF-B, nuclear factor-B; ATF-2, activated transcription factor 2; DC, dendritic cell; TBK, Tank-binding kinase 1; PKC, protein kinase C; IL, interleukin; ds--DNA, double-stranded -DNA; GFP, green fluorescent protein; ELISA, enzyme-linked immunosorbent assay; cPKC, conventional PKC.
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ACKNOWLEDGMENTS |
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and PKCI and Dr. Shizuo Akira (Department of Host Defense, Osaka, Japan) for generously supplying HEK 293 cells stably expressing TLR3. We also thank Drs. Stanislas Goriely and Annette Schoenemeyer for collaboration and Harlan Levey for critical review of the manuscript. |
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