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IFN Regulatory Factor-1 Is Required for the Up-Regulation of the CD40-NF-B Activator 1 Axis During Airway Inflammation¹

Zhendong Zhao^*, Youcun Qian^*, Dave Wald^*, Yi-Feng Xia, Jian-Guo Geng and Xiaoxia Li^2,*

^* Department of Immunology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH 44195; and Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

	Abstract

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Recent studies show that NF-B activator 1 (Act1) functions as an important adapter molecule for CD40-mediated signaling in epithelial cells. To explore the physiological function of the CD40-Act1 axis, we studied the regulation of gene expression of CD40 and Act1 both in vivo and in cell culture models. Although CD40 and Act1 are up-regulated in mouse lung upon LPS stimulation, IL-1 plus IFN-, -, or - synergistically up-regulate both CD40 and Act1 gene expression in human epithelial A549 cells. Cycloheximide superinduces the Act1 mRNA, whereas actinomycin D completely abolishes the Act1 mRNA, indicating that the induction of Act1 mRNA is at the transcriptional level and does not require protein synthesis. Promoter sequence analyses identified putative IFN regulatory factor (IRF)-1, C/EBP-, and AP-1 transcription factor binding sites in the Act1 promoter. Although mutation of any of the three sites abolished the promoter activity, Abs against IRF-1 and C/EBP-, but not AP-1, blocked the formation of the DNA-binding complex induced by IL-1 plus IFN- stimulation, suggesting cooperative action between IRF-1 and C/EBP- in mediating Act1 promoter activity. Importantly, LPS-induced gene expression of CD40 and Act1 in the mouse lung is abolished in IRF-1^-/- mice, indicating an essential role of transcription factor IRF-1 in the coordinated regulation of these two genes during airway inflammation. The induced expression of the CD40-Act1 axis by inflammatory cytokines in epithelial cells probably plays an important role in priming these cells for their response to CD40 ligand during airway inflammation.

	Introduction

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As a member of the TNFR superfamily, CD40 was first identified on B lymphocytes. The primary action of CD40-mediated pathway was thought to be restricted to thymus-dependent humoral responses (1). However, a variety of nonlymphocytic cell types have also been shown to express CD40, including monocytes, basophils, dendritic cells, fibroblasts, smooth muscle cells, endothelial cells, and epithelial cells (2, 3, 4, 5). Studies from many groups indicate that CD40 probably plays a more general role in immune regulation (6). CD40-CD40 ligand (CD40L)³ interactions have recently been implicated in airway inflammation (7, 8, 9, 10, 11, 12, 13, 14, 15). Although airway epithelial cells express low levels of CD40, inflammatory cytokines, including IL-1, TNF-, and IFN-, can all strongly up-regulate the expression of CD40 in airway epithelial cells (16, 17, 18). Ligation of CD40 on the airway epithelial cells up-regulates the expression of inflammatory mediators, including the chemokines IL-8, RANTES, and monocyte chemoattractant protein-1 and the adhesion molecule ICAM-1 (8). Using CD40L-null mice that were first sensitized and aerosol-challenged with OVA, studies showed the clear involvement of CD40-CD40L interaction in the development of allergic airway inflammation (10). After Ag challenge, sensitized control mice developed airway inflammation, including production of TNF- and IL-4 and expression of VCAM-1. This inflammatory response was dramatically reduced in CD40L-null mice (10). A chimeric mouse model based on CD40-null and wild-type mice was used to assess the contribution of bone marrow (BM)-derived and non-BM-derived cells in a CD40-mediated pulmonary inflammation response. It was found that while CD40 plus BM-derived cells are required for the initiation of inflammation, CD40 plus non-BM-derived cells are required to maximize the level of pulmonary inflammation, implicating a role for epithelial cells in airway inflammation (12). Taken together, these data suggest a role for CD40 in the regulation of inflammatory responses at mucosal sites.

The transcription factor NF-B is a central mediator of inflammatory responses. Although many stimuli lead to activation of NF-B, including cytokines, bacterial products, viruses, and environmental insults, the proximal signaling components of the pathways activated by most of these stimuli are still largely unknown. We recently studied CD40-mediated signaling pathway in epithelial cells. We found that a novel NF-B activator (Act1) plays a critical role in CD40-mediated NF-B activation in epithelial cells. Act1, a 60-kDa (574 aa) polypeptide, was cloned through a functional genetic screening based on its ability to activate NF-B (19). Simultaneously, Act1 was also cloned by Leonardi et al. (20), called CIKS (connection to I-B kinase and stress-activated protein kinase/Jun kinase), through a yeast two-hybrid screening, based on its interaction with I-B kinase-. Act1 does not have any enzymatic domain but does contain a helix-loop-helix at the N terminus, a coiled-coil at the C terminus, and two putative TNFR-associated factor (TRAF) binding sites, EEESE (residues 38–42) and EERPA (residues 333–337). Coimmunoprecipitation experiments revealed that Act1 strongly interacts with TRAF3 and weakly with TRAF1 and TRAF5 (21). TRAF3 and TRAF5 participate in signaling pathways mediated by CD40, lymphotoxin- receptor, and several other members of the TNFR superfamily. Interestingly, we found that endogenous Act1 indeed interacts with CD40, both in HeLa and HT29 cells stimulated with CD40L, strongly suggesting that Act1 plays a critical role in CD40-mediated signaling (21). Expression of Act1 in the natural Act1-null C33A cells renders these cells susceptible to CD40L-induced NF-B activation (21), showing that Act1 can mediate this process.

To understand the physiological function of the CD40-Act1 axis, in this report we studied the regulation of gene expression of CD40 and Act1 both in vivo and in cell culture models. We found that while Act1 and CD40 gene expression is induced in mouse lung upon LPS stimulation, their expression is also induced in lung epithelial A549 cells by inflammatory cytokines. The co-up-regulation of Act1 and CD40 in airway epithelial cells suggests that the CD40-Act1 axis probably plays an important role in airway inflammatory response.

	Materials and Methods

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Inflammatory mice model development and cell culture

Eight-week-old male IFN regulatory factor (IRF)-1^+/+ and IRF-1^-/- mice (The Jackson Laboratory, Bar Harbor, ME) were i.p. injected with LPS (Sigma-Aldrich, St. Louis, MO) at a dose of 100 µg/kg of mice weight. After 6 h of treatment, different tissues were dissected and subjected to RNA isolation. A549, a human epithelial carcinoma cell line, was maintained in MEM supplemented with 10% FCS, penicillin G (100 µg/ml), and streptomycin (100 µg/ml).

RNA isolation and Northern blot analysis

Total RNA from mice tissues and culture cells were extracted by TRIzol reagent (Life Technologies, Grand Island, NY). RNA samples were electrophoresed in 1% agarose-formaldehyde gels and transferred to Hybond-N⁺ membrane (Amersham, Arlington Heights, IL) in 10x SSC. DNA fragments used as probes were ³²P-labeled by random primer synthesis (Amersham). The blots were hybridized in Church’s buffer (0.5 M NaH₂PO₄, 0.5 M Na₂HPO₄, 0.5 M EDTA, 5% BSA, 7% SDS) at 65°C overnight. The blots were washed in 2x standard saline citrate phosphate-EDTA/0.1% SDS once and in 0.1x standard saline citrate phosphate-EDTA/0.1% SDS for 15 min twice at 65°C and exposed to Kodak X-OMAT AR films at -70°C with an intensifying screen.

5'-RACE analysis of Act1 and cloning of Act1 promoter

Total cellular RNA was isolated from 10⁷ A375 cells using TRIzol (Life Technologies). Total RNA (5 µg) was used for 5'-RACE analysis using 5'-Full RACE core set (Life Technologies) according to the manufacturer’s instructions. After the two-step PCR amplification, the fragments of different sizes were sequenced. The 990 bp of Act1 promoter region upstream from Act1 gene transcription start site was amplified from genomic DNA and cloned to pGL3-basic luciferase reporter vector (Promega, Madison, WI).

Transfection and luciferase assay

Luciferase reporter constructs containing various Act1 promoter mutants were transfected into A549 cells (2 x 10⁵ cells seeded on 6-well plates) using FuGENE (Roche Biomedical Laboratories, Burlington, NC). After 48 h, the cells were split into two 35-mm plates and stimulated with IL-1 and IFN- (for 24 h), and harvested and assayed for luciferase activity using the Enhanced Luciferase Assay kit (Promega).

Site-directed mutagenesis

Site-directed mutagenesis was performed by using a QuickChange mutagenesis kit (Stratagene, La Jolla, CA) as instructed by the manufacturer. The luciferase reporter construct containing the -200/+1 region of the Act1 promoter was used as the DNA template. The mutations in the binding sites for IRF-1 (TTTC changed to TCTG), C/EBP- (GAAA changed to GCAC), and AP-1 (TGAC changed to AGTC) were verified by sequence analysis.

Gel-shift assay

Oligo 1 (5'-AGGAGGTTTCTGTTTTAAGAAATAAAGTGA-3') and oligo 2 (5'-AGGAGGTTTCTGTTTTAAGAAATAAAGTGACTCCTCAGC-3') from the Act1 gene promoter were used as a probe. Complementary oligonucleotides, end-labeled with polynucleotide kinase (Boehringer Mannheim, Indianapolis, IN) and -³²P-labeled ATP, were annealed by slow cooling. Approximately 20,000 cpm of probe were used per assay. Cytoplasmic extracts were prepared as described by Kessler et al. (22). The binding reaction was conducted at room temperature for 20 min in a total volume of 20 µl containing 20 mM HEPES buffer, pH 7.0, 10 mM KCl, 0.1% Nonidet P-40, 0.5 mM DTT, 0.25 mM PMSF, and 10% glycerol. Anti-IRF-1 (C-20) and anti-c-fos (K-25) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-C/EBP- was a kind gift from Dr. W. Liao (M.D. Anderson Cancer Center, Houston, TX).

	Results

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Act1 and CD40 gene expression are induced in mouse lung upon LPS stimulation

We examined whether Act1 gene expression is regulated during inflammation and immune response. To induce inflammation, mice were i.p. injected with LPS. Northern blot analysis was performed to measure Act1 expression in different tissues of inflamed and control mouse. Act1 is highly expressed in mouse colon, moderately in heart, lung, spleen, and intestine, and expressed at very low levels in liver and brain. Interestingly, Act1 gene expression is induced in lung and intestine from an LPS-injected mouse as compared with a normal mouse (Fig. 1A). We used the chemokine macrophage-inflammatory protein-2 cDNA as a positive control for inflammation-induced gene expression. Macrophage-inflammatory protein-2 gene expression is highly induced in lung and heart and slightly induced in spleen, intestine, and colon.

View larger version (37K): [in this window] [in a new window]   FIGURE 1. Expression of Act1 and CD40 in mouse tissues. RNA from various mouse tissues with (+, 6 h) or without (-) LPS (10 µg/ml) stimulation and probed with mouse cDNAs of Act1 (A) and CD40 (B). rRNA (28s and 18s) or GAPDH were used to show equal loading.

Act1 and CD40 expression are induced in lung epithelial A549 cells by inflammatory cytokines

To study the regulation of Act1 gene expression in a cell culture model, we investigated whether Act1 mRNA can be induced in cultured cells in response to various inflammatory mediators. We first examined whether Act1 gene expression can be induced in mouse primary macrophages prepared by peritoneal lavage in response to LPS stimulation. Northern blot analysis showed that LPS induces the expression of TNF- but not Act1 in these macrophages (Fig. 2A). We then examined the Act1 gene expression in several human epithelial cell lines, including intestinal HT29, lung alveolar epithelial A549, keratinocyte HaCat, cervical carcinoma HeLa, and breast carcinoma MCF-7 cells, untreated or treated with inflammatory cytokines. Northern and Western blot analyses showed that the expression of Act1 in HT29, HeLa, HaCat, and MCF-7 cells is constitutively high (Fig. 2B), and further induction was not detected in these cell lines (data not shown). In contrast, while lung epithelial A549 cells have low basal Act1 gene expression, its expression is induced 2-fold by IL-1 and TNF-, 5-fold by LPS, and 20-fold by MLC-conditioned medium (CM) (Fig. 2, B and C). Treatment with IFN- or IFN- had no effect on the expression of Act1 in A549 cells (Fig. 2C and data not shown). With the same Northern blot, we also examined IL-8 and ICAM-1 gene expression in A549 cells in response to various stimulations. ICAM-1 gene expression is slightly induced by IFNs and LPS, but highly induced by IL-1/TNF- and CM. IL-8 gene expression is induced slightly by LPS, whereas it is highly induced by IL-1/TNF- and CM. The IL-8 gene expression induced by IL-1 and TNF- (20-fold) is much higher than its CM-induced expression (10-fold). The fact that CM-induced Act1 gene expression is higher than that induced by IL-1/TNF- suggests that additional cytokines in the CM are involved in the induction of Act1 gene expression. Pretreatment with cycloheximide (1 h) superinduces the Act1 mRNA upon stimulation with CM (4 h), whereas actinomycin D (pretreatment, 1 h) completely abolishes the Act1 mRNA, suggesting that the induction of Act1 mRNA by CM might be at the transcriptional level and does not require protein synthesis (Fig. 2D). Taken together, the above data show that Act1 is either constitutively high or its expression is induced in epithelial cells, suggesting that Act1 probably plays an important role in these cells.

View larger version (62K): [in this window] [in a new window]   FIGURE 2. Expression of Act1 mRNA. A, RNA from mouse primary macrophages untreated (-) or treated with LPS (1 µg/ml), probed with mouse Act1 and TNF- cDNAs. B, RNA from various human cell lines, probed with the Act1 cDNA. C, RNA from A549 cells untreated (-) or treated with IFNs (IFN- and IFN-, 1000 µ/ml), LPS (1 µg/ml), IL-1/TNF (IL-1 and TNF-, 10 ng/ml), and CM, probed with human Act1, ICAM-1, IL-8, and GAPDH cDNAs. D, RNA from A549 cells untreated (-), treated with CM for different times as indicated or pretreated with either actinomycin D (2.5 µg/ml) or cycloheximide (10 µg/ml) for 1 h followed by treatment with CM (4 h), probed with human Act1 cDNA.

View larger version (26K): [in this window] [in a new window]   FIGURE 3. Synergistic induction of Act1 mRNA by IL-1 plus IFN-. A, RNA from A549 cells untreated or stimulated with IL-1, IFN-, IL-1 + IFN-, and CM, probed with Act1 cDNA. B, RNA from A549 cells treated with IL-1 plus IFN- for different times as indicated, probed with Act1, ICAM-1, and GAPDH cDNAs. C, Extracts from A549 cells untreated (-) or treated with IL-1 plus IFN- for different times as indicated were immunoprecipitated with anti-Act1 and probed with anti-Act1. D, RNA from A549 cells treated with IL-1 + IFN- for different times as indicated and probed with CD40 and GAPDH cDNAs.

Cis elements of IRF-1, C/EBP-, and AP-1 are required for Act1 promoter activity

To further study the regulation of Act1 gene expression, we decided to clone the Act1 gene promoter. The transcriptional initiation site was determined by 5'-RACE analysis using RNAs from human malignant melanoma A375 cells (Figs. 4A and 5A). A 990-bp genomic fragment upstream of the transcriptional start site was amplified from the genomic DNA of A375 cells and cloned into the pGL3-basic luciferase reporter vector. To test the promoter activity, this Act1-Luc construct was transiently transfected into A549 cells, followed by stimulation with IL-1 plus IFN- or with CM and evaluation by luciferase reporter assay. As shown in Fig. 4B, while IL-1 plus IFN- induced the luciferase activity 3-fold, CM stimulation resulted in 5- to 7-fold induction, indicating that the Act1-Luc construct does contain a functional promoter. To identify the cis elements that are responsible for the induced promoter activity, deletion mutants of the Act1 5'-flanking region were generated in pGL3-basic vector. The resulting constructs were transiently transfected into A549 cells, treated with IL-1 plus IFN- or CM, and followed by luciferase reporter assay. We found that all the deletion mutants, including D200, can still drive signal-induced luciferase reporter expression (Fig. 4B), suggesting that the responsive cis elements might reside in this proximal 200-bp 5'-flanking region of Act1. Using Transfac program (Research Group Bioinformatics, Braunschweig, Germany), we identified IRF-1, C/EBP-, and AP-1 transcription factor binding sites in the Act1 promoter (Fig. 5A). To determine the functional importance of these sites, through site-directed mutagenesis we mutated the core-binding sequence of these transcription factor-binding sites (Fig. 5A). Interestingly, mutation of any of the three sites (IRF-1, C/EBP-, and AP-1) abolished the promoter activity, suggesting that these three sites are all necessary for the Act1 promoter activity (Fig. 5B). To identify the transcription factors that bind to the Act1 promoter, oligo 1 (containing the IRF-1 and C/EBP binding sites, Fig. 5A) and oligo 2 (containing IRF-1, C/EBP, and AP-1 binding sites, Fig. 5A) were subjected to gel-shift assay, using cell extracts prepared from A549 cells with or without IL-1 plus IFN- stimulation. As shown in Fig. 5C, the induced complex bound to oligo 1 (IRF-1 and C/EBP sites) was partially blocked by anti-IRF-1 and completely abolished by anti-C/EBP-, indicating the involvement of these two transcription factors in regulating the Act1 promoter activity. The induced complex bound to oligo 2 (IRF-1, C/EBP, and AP-1 sites) migrated similarly to that bound to oligo 1. Furthermore, while anti-IRF-1 and anti-C/EBP blocked complex formation on oligo 2, anti-c-fos and anti-jun had no effect (Fig. 5C and data not shown). Taken together, these results suggest that no additional factors were bound to the AP-1 site in oligo 2.

View larger version (39K): [in this window] [in a new window]   FIGURE 4. Deletion analysis of Act1 promoter. A549 cells transfected with various Act1 promoter deletion mutants were untreated or treated with IL-1 plus IFN- and CM for 4 h (A) followed by luciferase reporter assay (B). The data presented are the average from three experiments.

View larger version (25K): [in this window] [in a new window]   FIGURE 5. Analyses of the IRF-1, C/EBP-, and AP-1 binding sites in Act1 promoter. A, Act1 promoter sequence. The binding sites for IRF-1, C/EBP-, and AP-1 are underlined, and the mutated nucleotides are shown. The transcription start site is indicated. B, Luciferase reporter assay. Site-specific mutants of Act1 promoter were transfected into A549 cells and treated with IL-1 + IFN- for 4 h, followed by luciferase assay. The data presented are the average of three experiments. C, Gel-shift assay. Cell extracts were made from A549 cells untreated or treated with IL-1 + IFN- for 24 h. Oligo 1 and oligo 2 from the Act1 promoter (see Materials and Methods) were used as probes.

Previous studies suggested that IRF-1 might play a role in IFN--induced CD40 expression in endothelial cells. The above results indicate that IRF-1 is probably required for induced expression of Act1 in response to IL-1 plus IFN-. To investigate the role of transcription factor IRF-1 in the regulation of the CD40-Act1 axis during airway inflammation, we examined the expression of Act1 and CD40 in mouse lungs derived from IRF-1^-/- mice. To induce inflammation, control and IRF-1^-/- mice were i.p. injected with LPS, followed by Northern blot analysis to measure Act1 and CD40 expression in their lungs. While both Act1 and CD40 expression are induced in the lungs from IRF-1^+/+ mice upon LPS stimulation, the induced expression of Act1 and CD40 is abolished in IRF-1^-/- mice (Fig. 6). These results indicate that the transcription factor IRF-1 plays an essential role in the coordinated regulation of Act1 and CD40 during airway inflammation.

View larger version (36K): [in this window] [in a new window]   FIGURE 6. Expression of Act1 and CD40 in IRF-1-/- mice. RNA from IRF-1+/+ and IRF-1-/- lungs with (+, 6 h) or without (-) LPS (10 µg/ml) stimulation, probed with mouse cDNAs of Act1, CD40, and GAPDH.

	Discussion

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The analysis of the Act1 gene promoter reveals that transcription factors IRF-1, C/EBP-, and AP-1 are responsible for the induced expression of Act1 mediated by IL-1 and IFN-. Both C/EBP- and AP-1 binding sites have been identified in many IL-1-responsive gene promoters, indicating the role of these two transcription factors in mediating IL-1-induced gene expression (23). IL-1 stimulation leads to posttranslational modification of C/EBP- and AP-1, thereby increasing their DNA binding activity. IRF-1 plays an important role in IFN--, --, and --induced gene expression (24). Stimulation with IFNs leads to both increased expression of IRF-1 and its posttranslational modification, resulting in the activation of this transcription factor. In Act1 promoter, the binding site of IRF-1 overlaps with that of C/EBP-, followed immediately by the AP-1 binding site. Whereas mutation of any of the three sites abolished the promoter activity, Abs against IRF-1 and C/EBP-, but not AP-1, blocked the DNA binding complex formation on Act1 promoter upon IL-1 plus IFN- stimulation. Furthermore, while IL-1 or IFN- alone fail to induce the expression of Act1, IL-1 plus IFN- synergistically up-regulate Act1 gene expression. Taken together, these results strongly suggest that the induced expression of Act1 in response to IL-1 and IFN- is probably due to the cooperative action of the IRF-1 and C/EBP- binding sites in the Act1 promoter. Because we failed to detect any protein binding to the putative AP-1 site in the Act1 promoter, the transcription factor AP-1 is probably not a player in the regulation of Act1 gene expression.

Previous studies have shown that proinflammatory cytokines can up-regulate CD40 expression in various cell types. NF-B, STAT-1, and IRF-1 have been implicated in the regulation of CD40 expression. In this study, we have not only shown the interesting co-up-regulation of CD40 with its signaling molecule Act1 in mouse lung during airway inflammation, but also demonstrated that the induced expression of these two genes is abolished in IRF-1^-/- mice, indicating that IRF-1 is a common transcription factor required for the regulated expression of CD40 and Act1. The precise function of the CD40-Act1 axis in airway inflammation is still unclear. The identification of the target genes that are activated by this Act1-dependent CD40-mediated pathway will greatly facilitate the understanding of the functional role of the CD40-Act1 axis.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant GM 600020 (to X.L.).

² Address correspondence and reprint requests to Dr. Xiaoxia Li, Department of Immunology, NB30, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail address: Lix{at}ccf.org

³ Abbreviations used in this paper: CD40L, CD40 ligand; BM, bone marrow; Act1, NF-B activator 1; TRAF, TNFR-associated factor; CM, conditioned medium.

Received for publication October 29, 2002. Accepted for publication March 21, 2003.

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