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The Journal of Immunology, 2003, 171: 4187-4194.
Copyright © 2003 by The American Association of Immunologists

IFN-{gamma}-Induced MHC Class II Expression: Transactivation of Class II Transactivator Promoter IV by IFN Regulatory Factor-1 is Regulated by Protein Kinase C-{alpha} 1

Mélanie Giroux*, Manuel Schmidt{dagger} and Albert Descoteaux2,*

* Institut National de la Recherche Scientifique–Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada; and {dagger} Mologen GmbH, Berlin, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies based on pharmacological evidence suggested a requirement for protein kinase C (PKC) activity in the regulation of IFN-{gamma}-induced MHC class II (MHC-II) expression. In the present study, we investigated the molecular mechanisms by which PKC-{alpha} modulates IFN-{gamma}-induced MHC-II expression in the mouse macrophage cell line RAW 264.7. Overexpression of a dominant-negative (DN) mutant of PKC-{alpha} inhibited the expression of IFN-{gamma}-induced MHC-II but had no effect on IFN-{gamma}-induced STAT1 nuclear translocation and DNA binding activity, as well as on the expression of inducible NO synthase, IFN consensus sequence binding protein, MHC class I, IFN regulatory factor (IRF)-1, and IFN-{gamma}-inducible protein-10. Further analysis showed that IFN-{gamma}-induced expression of the MHC class II transactivator (CIITA), a transcriptional coactivator essential for MHC-II expression, was inhibited in DN PKC-{alpha}-overexpressing cells. Studies with reporter constructs containing the promoter IV region of CIITA revealed that overexpression of a constitutively active mutant of PKC-{alpha} enhanced IRF-1, but not IRF-2, transcriptional activity. Furthermore, characterization of IRF-1 from both normal and DN PKC-{alpha}-overexpressing cells revealed differences in IRF-1 posttranslational modifications. Collectively, our data suggest a novel regulatory mechanism for IFN-{gamma}-induced MHC-II expression, whereby PKC regulates CIITA expression by selectively modulating the transcriptional activity of IRF-1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Major histocompatibility complex class II (MHC-II) 3 molecules play a pivotal role in the induction and regulation of immune responses by virtue of their ability to present peptides derived mainly from processed extracellular Ags to CD4+ Th lymphocytes (1, 2). Although constitutively present on professional APCs (monocytes/macrophages, B cells, and dendritic cells), MHC-II expression can be induced in most cell types and tissues by IFN-{gamma} (1, 3, 4).

The regulation of both constitutive and IFN-{gamma}-inducible MHC-II expression occurs primarily at the transcriptional level. The signaling cascade leading to MHC-II gene expression in response to IFN-{gamma} is initiated by the activation of the protein tyrosine kinases JAK1 and JAK2 and the subsequent tyrosine phosphorylation and dimerization of STAT1. Then, STAT1 dimers translocate to the nucleus where they bind to IFN-{gamma}-activated sequence (GAS) elements present in the promoters of IFN-{gamma}-responsive genes (5). Such genes include the transcription factor IFN regulatory factor (IRF)-1 and the transcriptional coactivator class II transactivator (CIITA). IRF-1, which is strongly inducible by IFN-{gamma}, binds promoter sequences known as IFN-stimulated responsive element (ISRE) (6), and is essential for CIITA induction by IFN-{gamma} (7). CIITA is a non-DNA binding transcriptional coactivator essential for both constitutive and IFN-{gamma}-inducible MHC-II expression (8, 9). CIITA gene expression is controlled by the alternative usage of three distinct promoters: constitutive expression in dendritic cells and B lymphocytes by promoters I and III, respectively, and IFN-{gamma}-inducible expression in other cell types mainly by promoter IV (10). However, a recent study revealed that, in macrophages, both type I and type IV CIITA are responsive to IFN-{gamma} (11). Although the regulation of type I CIITA remains to be elucidated, IFN-{gamma} inducibility of type IV CIITA is dependent mainly upon an ISRE, a GAS, and an adjacent E-box, which bind the transcription factors IRF-1, STAT1, and upstream stimulating factor-1, respectively (12).

Previous studies using protein kinase C (PKC) inhibitors suggested that IFN-{gamma}-induced MHC-II expression requires the activation of PKC in macrophages and astrocytes (13, 14, 15). Furthermore, microinjection of peritoneal macrophages with purified PKC was sufficient to induce MHC-II expression, suggesting that PKC participates in the regulation of MHC-II expression (16). Despite considerable progress having been made concerning the elucidation of the transcriptional events leading to IFN-{gamma}-induced MHC-II expression, the role of PKC in this process remains poorly understood. At least six isoenzymes of PKC, a family of protein serine/threonine kinases, are expressed in macrophages, but knowledge of their respective roles in the regulation of macrophage functions is limited. Using clones of the RAW 264.7 macrophage cell line overexpressing a dominant-negative (DN) mutant of PKC-{alpha} (DN PKC-{alpha}) (17), we have accumulated evidence that PKC-{alpha} is involved in the regulation of several macrophage functions, including LPS- and IFN-{gamma}-induced responses, as well as phagocytosis (17, 18, 19). In the present study, we report that PKC-{alpha} selectively regulates IFN-{gamma}-induced expression of MHC-II by modulating the ability of IRF-1 to transactivate the CIITA promoter IV. These results suggest that PKC-{alpha} participates in the development of a proper adaptive immune response in macrophages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

The murine macrophage cell line RAW 264.7 transfected with the expression vector pCIN-4, and the DN PKC-{alpha}-overexpressing clones A2, B1, and C2 (17) were cultured in a 37°C incubator with 5% CO2 in DMEM with glutamine (Life Technologies, Burlington, Ontario, Canada), containing 10% heat-inactivated FBS (HyClone, Logan, UT), 10 mM HEPES (pH 7.3), antibiotics, and 500 µg/ml G418 (Life Technologies).

Northern blot analyses

Total RNA extraction and Northern blot analyses were performed essentially as described previously (17, 20). The probe for murine MHC-II (H2-IA-{alpha}; d haplotype) consisted of a 591-bp fragment from the MHC-II cDNA amplified by PCR using oligodeoxynucleotides AD-70 (forward) (5'-GGAATTCTGGGAATCTCAGGTTCCCAGTG-3') and AD-71 (reverse) (5'-GGAATTCTGAACACCATGCTCAGCCTCTG-3'). The probe for murine MHC class I (MHC-I) (H2-K d) consisted of a 518-bp fragment from the MHC-I cDNA amplified by PCR using oligodeoxynucleotides AD-92 (forward) (5'-GGAATTCGATTACATCGCCCTGAACG-3') and AD-93 (reverse) (5'-GGAATTCAATTCAAGGACAACCAGAACAGCAATG-3'). The probe for murine IFN-{gamma}-inducible protein-10 (IP-10) was the 500-bp EcoRI fragment from C7-1 (21) (American Type Culture Collection, Manassas, VA). The inducible NO synthase (iNOS) probe was the 817-bp HincII-EcoRI fragment from piNOSL3 (22) (provided by D. Radzioch (McGill University, Montreal, Canada)). The probe for murine IFN consensus sequence binding protein (ICSBP) was the 1.2-kb EcoRI fragment from pSK-ICSBP (23) (kindly provided by K. Ozato (National Institutes of Health, Bethesda, MD)). The probe for murine IRF-1 consisted of a 207-bp fragment from the IRF-1 cDNA obtained by PCR amplification using oligodeoxynucleotides AD-134 (forward) (5'-CAGAGGAAAGAGAGAAAGTCC-3') and AD-135 (reverse) (5'-CACACGGTGACAGTGCTGG-3').

RT-PCR

Total RNA was prepared as described previously (17, 20). The relative quantities of mRNA for CIITA and hypoxanthine guanine phosphoribosyltransferase (HPRT) were determined by RT-PCR. For cDNA synthesis, 5 µg of total RNA, 1 µl of oligo(dT) primer (Amersham Pharmacia Biotech, Piscataway, NJ), and RNase-free water were added to a sterile microcentrifuge tube to obtain a total volume of 14 µl. This reaction mixture was heated at 70°C for 10 min and placed on ice for 1 min, and the following components were added: 2 µl of 10x synthesis buffer (200 mM Tris-HCl (pH 8.4), 500 mM KCl, 25 mM MgCl2, and 1 µg/µl BSA), 1 µl of 10 mM dNTP mix (Amersham Pharmacia Biotech), 2 µl of 0.1 M DTT, and 1 µl of avian myeloblastosis virus reverse transcriptase (20 U/µl; Amersham Pharmacia Biotech). Samples were then incubated at room temperature for 10 min, 42°C for 50 min, and 70°C for 15 min. For PCR, samples were amplified under the following conditions: 30 s at 94°C, 1 min at 50°C, and 1 min at 72°C (30 cycles). After the PCR amplification reaction, 20 µl of PCR products were analyzed by electrophoresis in a 1.3% agarose gel. The primers used for CIITA were AD-157 (forward) (5'-CAAGTCCCTGAAGGATGTGGA-3') and AD-158 (reverse) (5'-ACGTCCATCACCCGGAGGGAC-3'). The primers used for type I CIITA were AD-263 (forward) (5'-AAGAGCTGCTCTCACGGGAAT-3') and AD-268 (reverse) (5'-CTCTGCTCCAATGTGCTCCTA-3'). The primers used for type IV CIITA were AD-267 (forward) (5'-ACAGCCACAGCCGCGACCATA-3') and AD-268 (reverse) (5'-CTCTGCTCCAATGTGCTCCTA-3'). The primers used for HPRT were AD-55 (forward) (5'-GTTGGATACAGGCCAGACTTTGTTG-3') and AD-56 (reverse) (5'-GATTCAACTTGCGCTCATCTTAGGC-3').

Western blot analyses

Western blot analyses were performed as described previously (17). Rabbit polyclonal antisera against IRF-1 and IRF-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Plasmids

For the dual luciferase assays, the upstream 377 bp of the type IV promoter of CIITA was PCR amplified and cloned into the pGL3-basic firefly luciferase reporter vector (Promega, Madison, WI). The two putative transcription factors binding sites, ISRE and GAS, were mutated as described previously (24). The GAS element was changed from TTCTGAGAAA to TAGTGAGAAA, and the ISRE motif was changed from GAAAGTGAAAGG to GAAAGTGGTGGG. Mouse IRF-2 expression vector was constructed as described (25). The primers used for PCR amplification of IRF-1 were as follows: forward, 5'-CGGATCCCTCCGGCACCCTCTGCGA-3', and reverse, 5'-GAATTCCGGAGGGAGACAAGAACGGGTCAGA-3'; and the IRF-1 product was then inserted into pcDNA3 (BamHI/EcoRI) (Invitrogen, NV Leek, The Netherlands). The constitutively active (CA) PKC-{alpha} expression vector was provided by Dr. G. Baier (University of Innsbruck, Innsbruck, Austria). The pRL-TK plasmid encoding the Renilla luciferase was from Promega. pRcCMV was obtained from Invitrogen.

Transient transfections

Adherent cells (2.5 x 105/well) were transfected using Fugene (Roche Diagnostics, Laval, Quebec, Canada) with 0.3 µg of the CIITA luciferase reporter construct, and various combinations of pRcCMV, CA PKC-{alpha}, IRF-1, and IRF-2 expression vector, as indicated in Results, for a total of 0.7 µg. All transfections included 0.15 µg of pRL-TK (Promega) to control for transfection efficiency. Cells were transfected with 100 µl of DNA/Fugene mix in 1 ml of medium for 6 h, and then fresh medium containing 5% heat-inactivated FBS was added. Cells were treated 18 h later with 100 U/ml IFN-{gamma} for 8 h and 30 min, and lysed in Reporter lysis buffer (Promega). Firefly and Renilla luciferase values were obtained by analyzing 20 µl of cell extracts with the Dual Luciferase kit (Promega) using a Lumat LB 9507 luminometer (EG & G Berthold, Nashua, NH). Statistically significant differences were identified using unpaired Student’s t test. Values of p = 0.05 were considered statistically significant.

Nuclear extracts and EMSA

Adherent cells (8 x 106/100-mm tissue culture dish) were stimulated with 100 U/ml IFN-{gamma} for the indicated time points, washed, and scraped into 1.5 ml of cold PBS. Cell suspensions were transferred into microcentrifuge tubes, pelleted, and the nuclear protein extracts were prepared essentially as described (25). Protein contents were determined using the BCA protein assay kit (Pierce, Rockford, IL) and the extracts were stored at -70°C. EMSA were performed using the following oligonucleotides. CIITA-IRF-1 consensus oligonucleotide (5'-GGTGTAGACAGAAAGTGAAAGGGGGAAAAGCGCCACAGA-3') corresponds to the type IV CIITA promoter sequence -81 to -43 (10, 12). The GAS probe (5'-AGCCATTTCCAGGAATCGAAA-3') was derived from the Mg21 promoter sequence and contains a GAS site identical with the optimum GAS sequence (TTCCSGGAA) for STAT-1 binding (26). 32P-Labeled CIITA-IRF-1 oligonucleotide was incubated with 10 µg of nuclear extracts for 30 min at room temperature in a volume of 20 µl containing 50 mM KCl, 2.5 mM MgCl2, 1 mM EDTA, 1 mM DTT, 10 mM Tris-HCl (pH 7.5), 12% glycerol, 1 µg of salmon sperm DNA, and 1 µg of poly(dI:dC). 32P-Labeled GAS probe was incubated with 5 µg of nuclear extracts for 20 min at room temperature in a volume of 20 µl containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 50 mM DTT, 5 mM MgCl2, 10% glycerol, 0.2% Nonidet P-40, and 1 µg of poly(dI:dC). The CIITA-IRF-1-protein or GAS-protein complexes were separated from free oligonucleotides by electrophoresis under nondenaturing conditions in a 6% polyacrylamide gel at 250 V in 1x TGE (50 mM Tris-HCl, 380 mM glycine, and 2 mM EDTA) and in a 5% polyacrylamide gel at 180 V in 0.5x TBE (44.5 mM Tris-HCl, 44.5 mM borate (pH 8.0), and 1 mM EDTA), respectively. The gels were exposed to a phosphor screen that was scanned on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Immunoprecipitations and immunoblotting

Adherent cells (8 x 106/100-mm tissue culture dish) were stimulated with 100 U/ml IFN-{gamma} for the indicated time points. Cells were washed once with PBS, homogenized in lysis buffer (10 mM Tris-HCl (pH 7.5), 1 mM EGTA, and 1% Triton X-100) containing protease and phosphatase inhibitors, and protein concentrations were determined using the BCA protein assay kit (Pierce). Immunoprecipitations and immunoblottings were performed as described previously (19). Anti-STAT1 or anti-JAK2 Abs were obtained from Upstate Biotechnology (Lake Placid, NY). Tyrosine phosphorylation was analyzed using the mouse anti-phosphotyrosine mAb (4G10).

In vivo phosphorylation of IRF-1

Adherent RAW 264.7 cells (1 x 107/100-mm tissue culture dish) were incubated for 2 h in 3 ml of phosphate-free DMEM with glutamine (Life Technologies) supplemented with 0.5 mCi/ml 32Pi (Amersham Biosciences, Baie d’Urfé, Quebec, Canada) before the addition of 100 U/ml IFN-{gamma} for the indicated time points. Cells were then washed with ice-cold PBS and homogenized in immunoprecipitation buffer containing protease and phosphatase inhibitors. Total extracts were immunoprecipitated as described previously (19) and separated on 10% SDS-PAGE. The gels were exposed to a phosphor screen that was scanned on a PhosphorImager (Molecular Dynamics) to determine IRF-1 phosphorylation.

Two-dimensional gel electrophoresis

Preparation of cell extracts and immunoprecipitation were performed as described previously (19). Anti-IRF-1 Abs used for immunoprecipitation were from Santa Cruz Biotechnology. Solubilization of proteins was performed in a buffer containing 8 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 40 mM dithioerythritol, 20 mM Tris-HCl, 2% immobilized pH gradient buffer (Amersham Biosciences), and 0.5 µl of bromophenol blue (0.5%). Proteins were first separated according to their isoelectrical point along 7-cm linear immobilized pH gradient strips (Amersham Biosciences) using IPGphor Isoelectric Focusing system (Amersham Pharmacia Biotech). The strips were then equilibrated in a solution containing 13 mM dithioerythritol for 10 min, followed by a solution containing 2.5% iodoacetamide for 5 min. The proteins were finally separated according to their molecular mass using 10% SDS-PAGE, transferred on Hybond-ECL membranes, and detected by Western blot analysis using anti-IRF-1 Abs (Santa Cruz Biotechnology).

Translocation of PKC-{alpha}

Adherent RAW 264.7 cells were transfected using Gene Porter (Gene Therapy Systems, San Diego, CA) with 0.6 µg of the human (h)PKC-{alpha}-GFP construct (provided by C. Quittau-Prévostel (Institut National de la Santé et de la Recherche Médicale, Montpellier, France)). Cells were seeded (1 x 105/well) in 24-well plates containing microscope coverslips (Fisher Scientific, Pittsburgh, PA) for 1 day, transfected with 250 µl of DNA/Gene Porter mix for 5 h, and further incubated in medium for 18 h. Cells were then stimulated for 2 h with 100 U/ml IFN-{gamma}, fixed with 2% formaldehyde, permeabilized with ice-cold PBS containing 0.1% Triton X-100 and 1% BSA for 10 min at room temperature, and then incubated with Alexa Fluor 568 phalloidin (Molecular Probes, Eugene, OR). Coverslips were washed six times with PBS and mounted with Fluoromount-G (Interscience, Markham, Ontario, Canada). Analyses were performed using a Bio-Rad Radiance 2000 confocal imaging system (Bio-Rad Laboratories, Hercules, CA) installed on an Eclipse E800 microscope (Nikon, Melville, NY). Phalloidin fluorescence and translocation of PKC-{alpha}-GFP were analyzed using an argon/krypton laser at 568 and 488 nm, respectively, both with a Plan Apo Nikon 60x (numerical aperture, 1.4) oil immersion lens. At least 20–30 cells from each of two independent experiments were examined under each experimental condition. Images were acquired in the normal scanning mode with a Kalman filter of 10 using the LaserSharp software (Bio-Rad Laboratories).

Interaction between PKC-{alpha} and IRF-1

293T cells were grown to 75–85% confluence in 100-mm tissue culture dish. Cells were transfected with 15 µg of IRF-1 expression vector, and either 15 µg of pRcCMV or CA PKC-{alpha} expression vector using the calcium phosphate coprecipitation technique. Plasmid DNA and water were mixed to a total volume of 450 µl, and 50 µl of 2.5 M CaCl2 was added. The calcium/DNA solution was mixed quickly with 500 µl of HeBS (2x) buffer (140 mM NaCl, 1.5 mM Na2HPO4, and 50 mM HEPES (pH 7.05)), and the precipitate was immediately added to the dishes. Cells were transfected 8 h at 37°C, washed twice with methionine-free DMEM with glutamine (Life Technologies), and then incubated for 16 h in methionine-free DMEM with glutamine supplemented with 0.2 mCi/ml [35S]methionine (Amersham Biosciences). After the labeling period, cells were washed with ice-cold PBS and then homogenized in lysis buffer (100 mM NaCl, 50 mM HEPES (pH 7.4), and 0.2% Triton X-100) containing protease and phosphatase inhibitors. Total cell extracts were subjected to immunoprecipitation as described previously (19) with Abs to IRF-1 (Santa Cruz Biotechnology) and to PKC-{alpha} (Santa Cruz Biotechnology). The IRF-1 immunoprecipitate was denatured by a treatment of 5 min at room temperature and 5 min at 95°C in 50 µl of elution buffer (1% SDS, 100 mM Tris-HCl (pH 7.4), and 10 mM DTT), and 10 µl of 10% BSA was added. Samples were then diluted in 1 ml with lysis buffer containing 10 mM iodoacetamide and cleared by centrifugation, and the second immunoprecipitation was performed with anti-IRF-1 and anti-PKC-{alpha} Abs. Proteins were analyzed on 10% SDS-PAGE, and dried gels were exposed to a phosphor screen that was scanned on a PhosphorImager (Molecular Dynamics).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DN PKC-{alpha} overexpression selectively inhibits IFN-{gamma}-induced MHC-II mRNA accumulation

Using clones of the mouse macrophage cell lines overexpressing a DN PKC-{alpha}, we previously obtained evidence for the involvement of PKC-{alpha} in the modulation of IFN-{gamma}-induced COX-2 expression in macrophages (18). To further investigate the role of PKC-{alpha} in the modulation of IFN-{gamma}-induced responses, we first determined whether IFN-{gamma} alters the intracellular localization of PKC-{alpha}. To this end, we transiently transfected RAW 264.7 cells with a PKC-{alpha}-GFP expression vector. Incubation in the presence of 100 U/ml IFN-{gamma} induced the translocation of PKC-{alpha}-GFP from the cytoplasm to the nucleus within 2 h (Fig. 1A). We next compared the expression of several genes in normal RAW 264.7 cells (transfected with the empty vector) and in DN PKC-{alpha}-overexpressing clones (B1 and C2) (17) after stimulation with 100 U/ml IFN-{gamma} alone or in combination with 100 ng/ml LPS. MHC-II mRNA accumulation was completely abrogated in DN PKC-{alpha}-overexpressing clones B1 and C2 (Fig. 1B, lanes 5 and 8) compared with the levels observed in normal cells (lane 2). It has been demonstrated that LPS down-modulates the accumulation of Ia mRNA induced by IFN-{gamma} (27, 28). Accumulation of MHC-II mRNA decreased in normal RAW 264.7 macrophages in response to a combination of IFN-{gamma} and LPS (Fig. 1B, lane 3) compared with the levels observed for IFN-{gamma}-induced mRNA in control cells (lane 2). Consistent with the inhibition of IFN-{gamma}-induced MHC-II mRNA accumulation, DN PKC-{alpha}-overexpressing clones B1 and C2 failed to expressed MHC-II mRNA in response to LPS and IFN-{gamma} (Fig. 1B, lanes 6 and 9). The inhibitory effect of DN PKC-{alpha} on IFN-{gamma}-induced MHC-II expression was not the consequence of a general defect in IFN-{gamma}-induced responses, because the expression of several other IFN-{gamma}-inducible genes (iNOS, ICSBP, IRF-1, MHC-I, and IP-10) was not affected by DN PKC-{alpha} (Fig. 1B, lanes 2, 5, and 8). Furthermore, IFN-{gamma}-induced tyrosine phosphorylation on STAT1 and JAK2 (Fig. 2A) as well as the kinetics of STAT1 nuclear translocation and DNA binding activity (B) were normal in DN PKC-{alpha}-overexpressing cells. Collectively, these data indicated that DN PKC-{alpha} selectively inhibits IFN-{gamma}-induced MHC-II mRNA accumulation, downstream of STAT1 activation.



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  		FIGURE 1. Effect of DN PKC-{alpha} overexpression on IFN-{gamma}-induced MHC-II mRNA accumulation. A, Adherent RAW 264.7 cells were transfected with hPKC-{alpha}-GFP, incubated with 100 U/ml IFN-{gamma} for 2 h, and prepared for confocal microscopy as described in Materials and Methods. hPKC-{alpha}-GFP is located in the cytoplasm of unstimulated cells and undergoes nuclear translocation following activation with 100 U/ml IFN-{gamma}. Similar results were obtained in two separate experiments. B, Adherent cells (vector alone, clone B1, and clone C2) were incubated in the absence (lanes 1, 4, and 7) or in the presence of either 100 U/ml IFN-{gamma} (lanes 2, 5, and 8) or a combination of 100 U/ml IFN-{gamma} and 100 ng/ml LPS (lanes 3, 6, and 9) for 8 h. For priming experiments, cells were first incubated with 100 U/ml IFN-{gamma} for 18 h followed by additional stimulation with IFN-{gamma}, or IFN-{gamma} and LPS. Total RNA was extracted, and Northern blot analysis was performed as described in Materials and Methods. Similar results were obtained in at least three separate experiments.

 


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  		FIGURE 2. PKC-{alpha} modulates IFN-{gamma}-induced CIITA transcriptional activity. A, Adherent cells (vector alone, clone B1, and clone C2) were incubated in the presence of 100 U/ml IFN-{gamma} for the indicated time points. Total RNA was extracted and subjected to RT-PCR with specific primers for CIITA and HPRT. B, Adherent cells (vector alone and clone C2) were incubated in the presence of 100 U/ml IFN-{gamma} for the indicated time points. Total RNA was extracted and subjected to RT-PCR with specific primers for type I and type IV CIITA and HPRT. C, Adherent RAW 264.7 cells were transiently transfected with the luciferase reporter vectors containing the normal, the GAS-mutated, and the ISRE-mutated type IV CIITA promoter region and the pRL-TK for 6 h along with either the control vector (pRcCMV) or the CA PKC-{alpha} (A25E) expression vector. Cells were incubated for 18 h and then stimulated with 100 U/ml IFN-{gamma} for 8 h and 30 min. Firefly and Renilla luciferase activities were determined in cell extracts. Data are expressed as a ratio of firefly luciferase value/Renilla luciferase value. Experiments were performed in triplicate and are representative of results obtained in three separate experiments. *, p <= 0.05 as compared with untreated or IFN-{gamma}-treated cells transfected with control vector.

 
PKC-{alpha} modulates IFN-{gamma}-induced type IV CIITA expression

CIITA is a master regulator of both inducible and constitutive MHC-II expression (7, 9). To elucidate the mechanism by which DN PKC-{alpha} inhibits IFN-{gamma}-induced MHC-II expression, we compared the accumulation of total CIITA mRNA in normal RAW 264.7 cells and in DN PKC-{alpha}-overexpressing clones B1 and C2 after stimulation with 100 U/ml IFN-{gamma}. In normal RAW 264.7 cells, IFN-{gamma} induced the expression of total CIITA mRNA in a time-dependent manner (Fig. 3A, lanes 1–4). In contrast, IFN-{gamma}-induced total CIITA mRNA levels were barely detectable in DN PKC-{alpha}-overexpressing clone B1 and C2 (Fig. 3A, lanes 5–12). Thus, similar to IFN-{gamma}-induced MHC-II expression, DN PKC-{alpha} overexpression inhibited IFN-{gamma}-induced CIITA gene expression in RAW 264.7 macrophages. These results raised the possibility that PKC-{alpha} modulates IFN-{gamma}-induced MHC-II gene expression by regulating CIITA expression. In macrophages, CIITA IFN-{gamma}-inducible expression is mainly regulated by promoters I and IV (10, 11, 29). In IFN-{gamma}-stimulated RAW 264.7 cells, type IV CIITA was induced earlier and more abundantly than type I CIITA (Fig. 3B, lanes 2 and 3). Overexpression of DN PKC-{alpha} strongly inhibited IFN-{gamma}-induced type IV CIITA mRNA accumulation and slightly reduced IFN-{gamma}-induced type I CIITA mRNA (Fig. 3B, lanes 5 and 6), suggesting that PKC-{alpha} modulates IFN-{gamma}-induced type IV CIITA expression. To further address the role of PKC-{alpha} in the induction of type IV CIITA expression, we transiently transfected RAW 264.7 cells with a CA PKC-{alpha} expression vector and a type IV CIITA promoter reporter construct. Expression of CA PKC-{alpha} had no significant effect on basal CIITA promoter IV activity in untreated RAW 264.7 cells (Fig. 3C). In contrast, CA PKC-{alpha} significantly increased IFN-{gamma}-stimulated CIITA promoter IV activity by ~1.6-fold (Fig. 3C; p = 0.005; n = 3) with respect to cells transfected with the control vector. Similar results were obtained with a wild-type PKC-{alpha} expression vector (data not shown). These data are consistent with PKC-{alpha} playing a role in modulating type IV CIITA expression in IFN-{gamma}-stimulated macrophages. To determine the influence of PKC-{alpha} on the various promoter IV elements, we transiently transfected RAW 264.7 cells with the CA PKC-{alpha} expression vector and reporter constructs containing either the GAS-mutated or the ISRE-mutated promoter IV region. As previously shown (12), we observed that both GAS and ISRE elements were important for promoter IV activity in untreated as well as in IFN-{gamma}-stimulated RAW 264.7 cells (Fig. 3C). In untreated cells, CA PKC-{alpha} significantly increased basal activity of promoter IV containing the GAS-mutated region by ~1.7-fold (Fig. 3C; p = 0.005; n = 3) with respect to cells transfected with the control vector. In IFN-{gamma}-treated cells, CA PKC-{alpha} significantly increased promoter activity of promoter IV containing the GAS-mutated region by ~2-fold (Fig. 3C; p = 0.002; n = 3) with respect to cells transfected with the control vector. But intriguingly, the construct containing the ISRE-mutated region was less induced by CA PKC-{alpha} in untreated and in IFN-{gamma}-treated cells. We obtained similar results with cells transfected with a wild-type PKC-{alpha} expression vector (data not shown). These results suggested that PKC-{alpha} might modulate the activity of ISRE-binding and IFN-{gamma}-activated factors to stimulate type IV CIITA promoter. These transcription factors may be IRF-1 or IRF-2



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  		FIGURE 3. Effect of DN PKC-{alpha} overexpression on IFN-{gamma}-induced activation of the JAK-STAT pathway. Adherent cells (vector alone, clone B1, and clone C2) were incubated in the presence of 100 U/ml IFN-{gamma} for the indicated time points. A, Total cell extracts were subjected to immunoprecipitation with anti-STAT1 and anti-JAK2 Abs, and then the levels of tyrosine phosphorylation were determined by Western blot analysis with anti-phosphotyrosine (4G10) mAb as described in Materials and Methods. B, Nuclear extracts were prepared, and EMSA for STAT1 were performed as described in Materials and Methods. In lane 13, 100-fold excess cold probe was coincubated with the reaction mixture containing nuclear extracts from control cells incubated with 100 U/ml IFN-{gamma} for 15 min before EMSA. Similar results were obtained in three different experiments.

 
Expression and DNA binding activity of IRF-1 and IRF-2 are normal in DN PKC-{alpha}-overexpressing RAW 264.7 cells

Because both IRF-1 and IRF-2 are involved in IFN-{gamma}-induced CIITA expression (7, 30), we have determined whether expression of these proteins was affected by DN PKC-{alpha}. In both control RAW 264.7 cells (Fig. 4A, lanes 1–4) and DN PKC-{alpha} overexpressing cells (clone B1, lanes 5–8; clone C2, lanes 9–12), IRF-1 was equally induced by IFN-{gamma}. Similarly, IRF-2 was constitutively expressed to similar levels in control cells (Fig. 4A, lanes 1–4) as well as in DN PKC-{alpha}-overexpressing clones B1 (lanes 5–8) and C2 (lanes 9–12). In addition, the kinetics of IRF-1 nuclear translocation and DNA binding activity were similar in IFN-{gamma}-stimulated control cells and DN PKC-{alpha}-overexpressing clones C2 (Fig. 4B). We obtained similar data with DN PKC-{alpha}-overexpressing clones B1 and A2 (data not shown). Supershift experiments, using Abs to IRF-1 and IRF-2, revealed that IRF-1 was the major factor bound to the IRF element in the CIITA promoter IV (data not shown). Collectively, these results indicated that IFN-{gamma}-induced IRF-1 expression and DNA binding activity, as well as IRF-2 expression, are not affected by DN PKC-{alpha}.



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  		FIGURE 4. Expression and DNA binding activity of IRF-1/2 are normal in DN PKC-{alpha}-overexpressing RAW 264.7 cells. A, Adherent cells (vector alone, clone B1, and clone C2) were incubated in the presence of 100 U/ml IFN-{gamma} for the indicated time points. Cell extracts were prepared for Western blot analyses as described in Materials and Methods. B, Adherent cells (vector alone, clone A2, clone B1, and clone C2) were incubated in the presence of 100 U/ml IFN-{gamma} for 1 and 8 h. Nuclear extracts were prepared, and EMSA for IRF-1 were performed as described in Materials and Methods. In lane 13, 100-fold excess cold probe was coincubated with the reaction mixture containing nuclear extracts from control cells incubated with 100 U/ml IFN-{gamma} for 1 h before EMSA. Similar results were obtained in three different experiments.

 
PKC-{alpha} increases IFN-{gamma}-induced transactivating activity of IRF-1, but not IRF-2

To investigate the role of PKC-{alpha} in IFN-{gamma}-induced activation of IRF-1 or IRF-2, we transiently transfected RAW 264.7 cells with CA PKC-{alpha}, IRF-1, or IRF-2 expression vectors, or a combination of CA PKC-{alpha} and IRF-1 or IRF-2 expression vectors along with the CIITA promoter IV reporter construct. As previously reported (30), we observed that expression of both IRF-1 and IRF-2 increased basal and IFN-{gamma}-induced promoter IV activity. In untreated RAW 264.7 cells, basal promoter IV activity was significantly increased by 4.2-fold with the IRF-1 expression vector (Fig. 5; p < 0.0001; n = 3) and by 3.9-fold with IRF-2 expression vector (Fig. 5; p < 0.0001; n = 3) with respect to cells transfected with the control vector. In IFN-{gamma}-treated RAW 264.7 cells, promoter IV activity was increased by 2.1-fold with the IRF-1 expression vector (Fig. 5; p < 0.0001; n = 3) and by 1.8-fold with the IRF-2 expression vector (Fig. 5; p = 0.004; n = 3) with respect to cells transfected with the control vector. Cotransfection of CA PKC-{alpha} with IRF-1 had a significant effect on IRF-1-transactivating activity, further increasing type promoter IV activity in untreated cells by 2-fold (Fig. 5; p < 0.0001; n = 3) and in IFN-{gamma}-treated cells by 1.4-fold (Fig. 5; p = 0.03; n = 3) with respect to cells transfected with the IRF-1 expression vector. In contrast, CA PKC-{alpha} had no effect on IRF-2-dependent basal (Fig. 5; p = 0.324; n = 3) and IFN-{gamma}-induced (Fig. 5; p = 0.843; n = 3) promoter IV activity. These data indicate that PKC-{alpha} modulates IRF-1- but not IRF-2-transactivating activity to induce CIITA promoter IV transcription.



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  		FIGURE 5. Overexpression of PKC-{alpha} increases IFN-{gamma}-induced transactivation activity of IRF-1 but not IRF-2. Adherent RAW 264.7 cells were transiently transfected with the CIITA promoter-IV/Luc reporter construct and pRL-TK for 6 h along with either the control vector (pRcCMV), the CA PKC-{alpha} (A25E) expression vector, and/or the IRF-1 or IRF-2 expression vector. Cells were incubated for 18 h and then stimulated with 100 U/ml IFN-{gamma} for 8 h and 30 min. Firefly and Renilla luciferase activities were determined in cell extracts. Data are expressed as a ratio of firefly luciferase value/Renilla luciferase value. Experiments were performed in triplicate and are representative of results obtained in three separate experiments. *, p <= 0.05 as compared with untreated or IFN-{gamma}-treated cells transfected with control vector. **, p <= 0.05 as compared with untreated or IFN-{gamma}-treated cells transfected with IRF-1 expression vector.

 
Effect of DN PKC-{alpha} overexpression on the modulation of IFN-{gamma}-induced IRF-1 posttranslational modifications

Both IRF-1 and IRF-2 undergo posttranslational modifications such as phosphorylation and acetylation (31, 32, 33, 34, 35). To assess the impact of PKC-{alpha} on IRF-1 posttranslational modifications, we first compared the phosphorylation of IRF-1 in IFN-{gamma}-treated normal RAW 264.7 cells and DN PKC-{alpha}-overexpressing cells. As shown in Fig. 6A, there were no detectable differences in the kinetics and levels of IRF-1 phosphorylation in these cells. However, two-dimensional gel electrophoresis analysis revealed that the migration in a pH gradient of IRF-1 from DN PKC-{alpha}-overexpressing cells was delayed with respect to the migration of IRF-1 from normal RAW 264.7 cells (Fig. 6B). To determine whether IRF-1 and PKC-{alpha} directly interact, we performed an immunoprecipitation-recapture assay. The observation that PKC-{alpha} was recaptured from IRF-1 immunoprecipitate in 293T cells transfected with both IRF-1 and CA PKC-{alpha} expression vectors (Fig. 6C, lane 7) suggested that both proteins interact in vivo. The specificity of the interaction was evidenced by the absence of bands corresponding to PKC-{alpha} in IRF-1 immunoprecipitate from cells transfected only with the IRF-1 expression vector (Fig. 6C, lane 3).



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  		FIGURE 6. Role of PKC-{alpha} in the modulation of IFN-{gamma}-induced IRF-1 posttranslational modifications. A, Adherent cells (vector alone and clone C2) were incubated in 32Pi-containing medium for 2 h before the addition of 100 U/ml IFN-{gamma} for the indicated time points. Immunoprecipitation of IRF-1 was carried as described in Materials and Methods. B, Adherent cells (vector alone and clone C2) were treated with 100 U/ml IFN-{gamma} for 1 h. Total cell lysates were immunoprecipitated with anti-IRF-1 Abs, and two-dimensional gel electrophoresis was performed as described in Materials and Methods. IEF, Isoelectric focusing. C, 293T cells were transiently transfected with an IRF-1 expression vector, together with either pRcCMV or a CA PKC-{alpha} expression vector for 8 h, followed by an incubation in [35S]methionine-containing medium for 16 h. Total cell extracts were prepared as described in Materials and Methods and subjected to immunoprecipitation with anti-IRF-1 (lanes 1 and 5) and anti-PKC-{alpha} (lanes 4 and 8) Abs. The IRF-1 immunoprecipitate was denatured as described in Materials and Methods. After dilution with lysis buffer, aliquots were subjected to reprecipitation with Abs to IRF-1 (lanes 2 and 6) and PKC-{alpha} (lanes 3 and 7). Proteins were resolved on a 7.5% SDS-PAGE, and the gel was dried and exposed on a phosphor screen that was scanned on a PhosphorImager. The presence PKC-{alpha} in lane 7 indicates that it was coprecipitated with IRF-1. The amount of IRF-1 immunoprecipitate loaded in lanes 1 and 5 represents one-tenth of the total sample. Data shown are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies based on pharmocological agents suggested that PKC participates in IFN-{gamma}-induced MHC-II expression in various cell types, including macrophages (13, 14, 36, 37). Our data obtained with DN PKC-{alpha}-overexpressing cells revealed a role for PKC-{alpha} in the regulation of MHC-II expression in IFN-{gamma}-treated RAW 264.7 macrophages. Significantly, overexpression of DN PKC-{alpha} had no effect on IFN-{gamma}-induced expression of several unrelated genes (iNOS, IRF-1, IP-10, ICSBP, and MHC-I), supporting the notion that PKC-{alpha} selectively modulates IFN-{gamma}-induced responses.

The inhibition of IFN-{gamma}-induced CIITA mRNA accumulation observed in DN PKC-{alpha}-overexpressing cells indicated that PKC-{alpha} regulates MHC II expression at the level of CIITA expression. This role for PKC-{alpha} was further substantiated by the fact that expression of a CA PKC-{alpha} significantly increased IFN-{gamma}-induced CIITA promoter IV activity in transient transfection assays. Dissection of the IFN-{gamma}-induced signaling cascade leading to CIITA expression revealed that kinetics and extent of tyrosine phosphorylation of both STAT1 and JAK2 were similar in control RAW 264.7 cells and in DN PKC-{alpha}-overexpressing cells. Furthermore, nuclear translocation and DNA-binding activity of STAT1 took place normally in DN PKC-{alpha}-overexpressing cells. These data indicated that the inhibitory effect of DN PKC-{alpha} on IFN-{gamma}-induced CIITA expression may be downstream of these early signaling events. This was expected because IFN-{gamma}-induced expression of iNOS, MHC I, ICSBP, and IRF-1 was normal in DN PKC-{alpha}-overexpressing RAW 264.7 cells. The observation that DN PKC-{alpha} had no effect on both the IFN-{gamma}-induced IRF-1 expression and IRF-1 DNA binding activity raised the possibility that PKC-{alpha} modulates IRF-1-transactivating capacity. Indeed, expression of a CA PKC-{alpha} enhanced the ability of IRF-1, but not IRF-2, to transactivate CIITA promoter IV in both unstimulated and IFN-{gamma}-treated RAW 264.7 cells, thereby strongly supporting this view. Although the underlying mechanism is presently unknown, there is growing evidence that phosphorylation is important for IRF-1 function (31, 38). The possibility that PKC-{alpha} participates to posttranslational modifications of IRF-1 is supported by the finding that the IRF-1 protein complex is more basic in DN PKC-{alpha}-overexpressing cells than in control cells and by the observation of a direct interaction between PKC-{alpha} and IRF-1. Mutation analysis of serine and threonine residues potentially phosphorylated by PKC will be required to elucidate the mechanism by which PKC-{alpha} modulates IRF-1 activity.

Studies with cells from mice with a targeted disruption of the IRF-1 gene established that IRF-1 is essential for IFN-{gamma}-induced expression of a number of genes including CIITA, iNOS, and MHC-I (7, 39, 40, 41). The observation that IFN-{gamma}-induced expression of iNOS and MHC-I was normal in DN PKC-{alpha}-overexpressing cells suggests that PKC-{alpha}-mediated modulation of IRF-1 activity may not be required for the transactivation of all IRF-1-dependent promoters but rather that this requirement is limited to a subset of promoters. The expression of a given gene depends on the simultaneous interactions of a specific combination of regulatory proteins within the control DNA elements, leading to the assembly of nucleoprotein structures termed enhanceosomes (42, 43). It is possible that, in the context of CIITA promoter IV, PKC-{alpha}-dependent posttranslational modification(s) of IRF-1 may be required for the stability of the enhanceosome or for the presentation of a specific activation surface. Whole genome expression analysis in DN PKC-{alpha}-overexpressing RAW 264.7 cells may identify genes other than CIITA whose expression requires PKC-{alpha}-dependent modulation of IRF-1-transactivating activity.

The inhibition of IFN-{gamma}-mediated induction of CIITA and MHC II expression by DN PKC-{alpha} indicates that PKC-{alpha} activation is an important event in IFN-{gamma}-induced expression of these genes in RAW 264.7 macrophages. However, the mechanism by which PKC-{alpha} is activated in IFN-{gamma}-treated macrophages remains to be established. PKC-{alpha} is activated by diacylglycerol which can be generated through different pathways, including the activation of phospholipase C. In the human epithelial cell line NCI-H292, IFN-{gamma}-induced ICAM-1 expression involves a c-Src tyrosine kinase activation pathway in which phospholipase C-{gamma}2 and PKC-{alpha} play an important role (44). Additional studies will be required to determine whether a similar pathway is involved in the activation of PKC-{alpha} in IFN-{gamma}-treated macrophages.

The JAK-STAT signaling pathway plays a central role in IFN-{gamma}-induced responses (5). Recent evidence indicate that members of the PKC family are required for proper function and fine tuning of this pathway. Hence, PKC-{delta} mediates serine phosphorylation of STAT1 to facilitate transcription of IFN-{gamma}-stimulated genes (45), whereas PKC-{epsilon} is required for optimal JAK-mediated STAT1 tyrosine phosphorylation (46). This suggests that both PKC-{delta} and PKC-{epsilon} have a broad impact on IFN-{gamma} responses. In contrast, PKC-{alpha} acts further downstream by modulating the transcriptional activity of IRF-1 in the context of CIITA promoter IV. In this respect, PKC-{alpha} appears to exert a narrower influence than PKC-{delta} and PKC-{epsilon} in the regulation of IFN-{gamma}-induced responses. Collectively, these data support the view that PKC isoenzymes modulate specific signaling and transcriptional events in IFN-{gamma}-stimulated cells.


   Acknowledgments
 
We are grateful to Dr. G. Baier for providing the CA PKC-{alpha} expression vector and Dr. C. Quittau-Prévostel for providing the PKC-{alpha}-GFP expression vector. We thank M. Desrosiers for his technical expertise in confocal microscopy.


   Footnotes
 
1 This work was supported by Grant MT-12933 from the Canadian Institutes of Health Research (to A.D.), the Deutsche Jose-Carreras-Leukämiestiftung e.V. (to M.S.), and the Wilhelm Sander-Stiftung (to M.S.). M.G. is the recipient of a studentship from the Fonds de la Recherche en Santé du Québec. A.D. is chercheur-boursier of the Fonds de la Recherche en Santé du Québec and the holder of a Canada Research Chair. Back

2 Address correspondence and reprint requests to Dr. Albert Descoteaux, Institut National de la Recherche Scientifique–Institut Armand-Frappier, 531 Boulevard des Prairies, Laval, Québec, Canada H7V 1B7. E-mail address: albert.descoteaux{at}inrs-iaf.uquebec.ca Back

3 Abbreviations used in this paper: MHC-II, MHC class II; MHC-I, MHC class I; GAS, IFN-{gamma}-activated sequence; IRF, IFN regulatory factor; CIITA, class II transactivator; ISRE, IFN-stimulated responsive element; PKC, protein kinase C; DN, dominant negative; IP-10, IFN-{gamma}-inducible protein-10; iNOS, inducible NO synthase; ICSBP, IFN consensus sequence binding protein; HPRT, hypoxanthine guanine phosphoribosyltransferase; CA, constitutively active; h, human. Back

Received for publication May 1, 2003. Accepted for publication August 5, 2003.


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 Results
 Discussion
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