J Biol Chem, Vol. 273, Issue 49, 32670-32678, December 4, 1998

H₂O₂ and Tumor Necrosis Factor- Induce Differential Binding of the Redox-responsive Transcription Factors AP-1 and NF-B to the Interleukin-8 Promoter in Endothelial and Epithelial Cells^*

Venkatesh Lakshminarayanan, Elizabeth A. Drab-Weiss^§, and Kenneth A. Roebuck^¶

From the Department of Immunology/Microbiology, Rush-Presbyterian, St. Luke's Medical Center, Chicago, Illinois 60612

	ABSTRACT

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We previously demonstrated that tumor necrosis factor- (TNF) and H₂O₂ differentially regulate interleukin-8 (IL-8) and intercellular adhesion molecule (ICAM-1) gene expression in endothelial and epithelial cells. H₂O₂ induced IL-8 expression in the A549 and BEAS-2B epithelial cell lines, but not in the human microvessel endothelial cell line, HMEC-1 or human umbilical vein endothelial cells. In contrast, H₂O₂ induced ICAM-1 only in endothelial cells. Unlike H₂O₂, the proinflammatory cytokine TNF induced IL-8 and ICAM-1 in both cell types. In this study, we examine the role of the redox-responsive transcription factors AP-1 and nuclear factor-B (NF-B) in the differential expression of IL-8. DNA binding studies using nuclear protein extracts from HMEC-1 and A549 cells stimulated with H₂O₂ or TNF demonstrated differential activation and promoter binding of AP-1 and NF-B. H₂O₂ activated AP-1 but not NF-B in A549, whereas TNF activated AP-1 as well as NF-B. In HMEC-1, TNF activated NF-B but not AP-1, while H₂O₂ did not activate either transcription factor. The differential activation of the factors was also reflected in their differential binding to the IL-8 promoter. Moreover, the H₂O₂ concentration dependent increase in epithelial IL-8 mRNA expression directly corresponded to the H₂O₂ concentration dependent binding of AP-1 to the IL-8 promoter. Supershift analysis revealed H₂O₂ as well as TNF induced AP-1 complexes containing c-Fos and JunD. TNF induced NF-B complexes containing Rel A (p65). Immunohistochemical staining of HMEC-1 and A549 cells revealed TNF stimulated nuclear localization of Rel A, whereas no translocation of Rel A was detected in either cell type stimulated by H₂O₂. These data indicate that the cell type-specific induction of IL-8 gene expression by H₂O₂ and TNF in HMEC-1 and A549 cells can be explained by the differential binding of AP-1 and NF-B to the IL-8 promoter.

	INTRODUCTION

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The chemotactic factor, interleukin-8 (IL-8),¹ a member of the CXC chemokine family (1), is a potent activator and chemoattractant of neutrophils (2) and is secreted as a 72- or 77-amino acid protein by a wide variety of cell types including endothelial and epithelial cells (3, 4). IL-8 is induced by the proinflammatory cytokines TNF, IL-1, IL-6, and interferon- (3, 5-7), and inhibited by the anti-inflammatory cytokine IL-10 (8, 9). IL-8 is regulated primarily at the level of gene transcription (10-13) and its promoter region contains functional binding sites for the transcription factors NF-B, C/EBP, and AP-1 (14-16). TNF activates the IL-8 and ICAM-1 genes through a cooperative interaction between NF-B and C/EBP binding to a composite enhancer element within the proximal promoter (7, 17-19).

IL-8 is induced by oxidant stress (20, 21), and antioxidants have been shown to inhibit IL-8 expression (22, 23). H₂O₂ increases IL-8 expression in epithelial cell lines, fibroblasts, and whole blood (20, 21), and hypoxia followed by reoxygenation increases IL-8 expression in mononuclear and endothelial cells (24, 25) and in the lung and myocardium in vivo (26, 27). Nitric oxide, a reactive nitrogen species, has been shown to induce IL-8 through the activation of NF-B (28).

We recently reported that H₂O₂ and TNF differentially regulate IL-8 and ICAM-1 gene expression in epithelial and endothelial cells (20). IL-8 was induced by H₂O₂ in epithelial cells but not in endothelial cells. In contrast, H₂O₂ induced ICAM-1 in endothelial cells but not in epithelial cells. TNF has also been shown to generate oxidant stress (29, 30) and induce IL-8 and ICAM-1 expression in both cell types (20). In addition, TNF and H₂O₂ induce a differential pattern of CC chemokine expression in epithelial cells. While TNF induced both RANTES and MCP-1, H₂O₂ only induced MCP-1 in A549 cells (20). These studies suggest that oxidant stress constitute cell type- and gene type-specific activation signals in epithelial and endothelial cells that may critically influence the site-specific recruitment of leukocyte subsets in inflammatory reactions.

The aim of the present study was to investigate the transcriptional mechanism by which H₂O₂ and TNF differentially regulate IL-8 gene expression in endothelial and epithelial cells. We demonstrate that the redox-sensitive transcription factors AP-1 and NF-B are differentially activated by H₂O₂ and TNF in HMEC-1 and A549 cells. Our findings suggest that the discordant binding of the redox-responsive transcription factors AP-1 and NF-B to the IL-8 gene promoter mediate the distinct pattern of IL-8 observed in epithelial and endothelial cells.

	EXPERIMENTAL PROCEDURES

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Materials-- 30% H₂O₂, MOPS, fibronectin, fetal bovine serum, diamide, and 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide were purchased from Sigma. Dulbecco's modified Eagle's medium, antimyotics, 10 × phosphate-buffered saline (PBS), F-12K media, MCDB-131 media, and 1% trypsin-EDTA were purchased from Life Technologies, Inc. (Grand Island, NY). Antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). [-³²P]ATP (3000 Ci/mmol) was purchased from NEN Life Science Products Inc. (Boston, MA). The IL-8 enzyme-linked immunosorbent assay kit was purchased from BioSource (Camarillo, CA). The A549 cell line was obtained from American Type Culture Collection (Rockville, MD). The human microvessel endothelial cell line (HMEC-1) was obtained from the Center for Disease Control (Atlanta, GA). Human epidermal growth factor was purchased from Becton Dickenson (San Jose, CA).

Cell Culture and Treatments-- The A549 human type II lung carcinoma cell line was grown and maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum, 1% penicillin/streptomycin, and 1% gentamycin to 90% confluence. HMEC-1 was cultured in MCDB-131 media (Life Technologies, Inc.) with 10% fetal calf serum, 1% penicillin/streptomycin, 1% gentamycin, 1 µg/ml hydrocortisone, and 0.01 µg/ml epidermal growth factor to 90% confluence in 24-well dishes. Cells were washed twice with 1 × PBS and covered with serum-free, phenol red-free and growth factor-free media for 24 h prior to agonist treatment. H₂O₂ and TNF were diluted to final concentration in serum and growth factor-free, phenol red-free media and applied to cells as indicated in the figure legends.

DNA Binding Studies-- Electrophoretic mobility shift assays (EMSA) were performed essentially as described (13). Briefly, nuclear protein extracts (3-6 µg of protein) prepared from A549 or HMEC-1 cells by the method of Osborn et al. (31) were incubated with 50,000 cpm (~0.1 ng) of ³²P-end-labeled oligonucleotide probes listed in Table I for 20-30 min at room temperature in 10 or 20 µl reaction volumes containing 12% glycerol, 12 mM HEPES-NaOH (pH 7.9), 60 mM KCl, 5 mM MgCl_2, 4 mM Tris-Cl (pH 7.9), 0.6 mM EDTA (pH 7.9), 0.6 mM dithiothreitol, and 0.25 µg of poly(dI-dC). To demonstrate binding specificity, 100-fold molar excess (10 ng) of a specific or nonspecific oligonucleotide as indicated in the figure legends was included in the binding reaction. For supershift analysis, antibodies (1-3 µg) against AP-1 (c-Jun, c-Fos, JunD) or NF-B (Rel A, p50, c-Rel) subunits (Santa Cruz Biotechology, Santa Cruz CA) were included in the binding reaction. Protein-DNA and protein-DNA-antibody complexes were resolved in 5% polyacrylamide gels pre-electrophoresed for 30 min at room temperature in 0.25 × TBE buffer (22.5 mM Tris borate and 0.5 mM EDTA, pH 8.3). Gels were dried and exposed to radiographic film with an intensifying screen at 70 °C. Gel shifts were performed at least twice with using nuclear extract prepared from different batches of cells. Similar results were obtained and a representative gel is shown in the figures.

Immunostaining-- A549 and HMEC-1 were grown on coverslips to 80% confluence. Cells were washed twice in PBS and fixed in 3.7% formaldehyde (diluted in PBS containing 0.2% Triton X-100) for 10 min. The coverslips were washed three times in PBS and covered with 0.2% bovine serum albumin diluted in PBS (blocking buffer) for 10 min. The blocking buffer was removed by draining and 4-8 µg of anti-human NF-B (Rel A/p65) antibody (rabbit polyclonal IgG purchased from Upstate Biotechnology, NY) diluted in 100 µl of blocking buffer added to the cells for 60 min. The antibody was removed and the cells washed three times in PBS. The cells were then incubated with the secondary antibody fluorescein-tagged goat anti-rabbit IgG. After 60 min, the secondary antibody was removed and the cells washed three time in PBS. The coverslips were mounted to slides containing Citifluor. Immunofluorescent Rel A staining was detected using a fluorescent microscope.

	RESULTS

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H₂O₂ and TNF Induce Different Patterns of AP-1 and NF-B Binding Activity in HMEC-1 and A549-- To investigate the role of the redox-sensitive transcription factors AP-1 and NF-B in the cell type-specific expression of IL-8 gene expression, we first performed a series of DNA binding studies using consensus binding sites for AP-1 and NF-B (Table I). Nuclear protein extracts were prepared from A549 epithelial cells and HMEC-1 endothelial cells exposed for 15, 30, or 60 min to concentrations of H₂O₂ or TNF shown previously to activate IL-8 gene expression. DNA binding activity was determined by the EMSA. As shown in Fig. 1, H₂O₂ increased AP-1 binding activity in A549 (Fig. 1A) but not NF-B binding activity (Fig. 1B). AP-1 binding activity was detected as early as 15 min and was increased at 60 min (Fig. 1A). In contrast, TNF increased both AP-1 and NF-B binding activity in A549 cells, although their kinetics of induction differed. AP-1 binding activity induced by TNF peaked at 30 min (Fig. 1A), whereas NF-B binding activity continued to increase over the 60-min time course (Fig. 1B).

View larger version (51K): [in this window] [in a new window]   Fig. 1.   H2O2 and TNF induce different patterns of AP-1 and NF-B binding activity in A549. Nuclear protein extracts from A549 cells treated with H2O2 (800 µM) or TNF (100 units/ml) for 15, 30, and 60 min were incubated with 32P-end-labeled consensus AP-1-binding site oligonucleotide (A) or consensus NF-B-binding site oligonucleotides (B). Gel shift complexes were resolved by electrophoresis and visualized by autoradiography. Specificity of the induced complexes was determined by competition with a 100-fold molar excess of unlabeled cAP-1 or cNF-B oligonucleotide. Arrows indicate migration of the induced DNA binding complexes. NIC indicates the migration of the non-induced nonspecific gel shift complexes. The migration of the free probe is not shown.

In contrast to A549, HMEC-1 displayed a very different pattern of binding activity for the two transcription factors. As shown in Fig. 2, H₂O₂ and TNF did not increase AP-1 binding activity over the constitutive binding activity in HMEC-1 and in fact slightly reduced this constitutive binding activity at 15 and 30 min (Fig. 2A). However, like A549, TNF induced NF-B binding activity in HMEC-1 and the kinetics were similar in the two cell types (Fig. 2B). NF-B binding activity was detected at 15 min and continued to increase at 30 and 60 min. These data indicate that H₂O₂ and TNF differentially activate the redox-responsive transcription factors AP-1 and NF-B in A549 and HMEC-1 providing a potential mechanism for the cell type-specific expression of IL-8 and ICAM-1 in epithelial and endothelial cells we previously reported (13, 20).

View larger version (57K): [in this window] [in a new window]   Fig. 2.   H2O2 and TNF induce different patterns of AP-1 and NF-B binding activity in HMEC-1. Nuclear protein extracts from HMEC-1 cells treated with H2O2 (100 µM) or TNF (100 units/ml) for 15, 30, and 60 min were incubated with 32P-end-labeled consensus AP-1-binding site oligonucleotide (A) or consensus NF-B-binding site oligonucleotide (B). Gel shift complexes were resolved by electrophoresis and visualized by autoradiography. Specificity of the induced complexes was determined by competition with a 100-fold molar excess of unlabeled cAP-1 or cNF-B oligonucleotide. Arrows indicate migration of the induced DNA binding complexes. NIC indicates the migration of the non-induced nonspecific gel shift complexes. The migration of the free probe is not shown.

H₂O₂ and TNF Induce Different Patterns of AP-1 and NF-B Binding Activity to the IL-8 Promoter in A549 and HMEC-1-- To determine whether the differential activation of AP-1 and NF-B could mediate the cell type-specific expression of IL-8, we examined the AP-1 and NF-B binding activities on the IL-8 promoter. Oligonucleotide sequences corresponding to the AP-1 and NF-B-binding sites of the IL-8 promoter (Table I) were assessed for binding activity by EMSA. As shown in Figs. 3 and 4, H₂O₂ and TNF induced different patterns of binding to the IL-8 promoter sites. H₂O₂ induced AP-1 binding activity on the IL-8 promoter in A549 (Fig. 3A), whereas in HMEC-1 AP-1 binding activity was constitutive (Fig. 4A). In contrast to H₂O₂, TNF induced NF-B binding activity in both cell types (Figs. 3B and 4B). TNF also induced AP-1 binding in A549 cells (Fig. 3A). Thus, the same pattern and kinetics of binding of AP-1 and NF-B to the consensus sites was observed on the IL-8 promoter sites. The correspondence between the consensus and IL-8-binding sites suggests that the distinct expression patterns of IL-8 induced by H₂O₂ and TNF in epithelial and endothelial cells is mediated by the differential activation of AP-1 and NF-B binding to the IL-8 promoter.

View larger version (50K): [in this window] [in a new window]   Fig. 3.   H2O2 and TNF induce different patterns of AP-1 and NF-B binding activity on the IL-8 promoter in A549 cells. Nuclear protein extracts from A549 cells treated with H2O2 (800 µM) or TNF (100 units/ml) for 15, 30, and 60 min were incubated with 32P-end-labeled oligonucleotides from the AP-1-binding site in the IL-8 promoter (A) or the NF-B-binding site in the IL-8 promoter (B). Gel shift complexes were resolved by electrophoresis and visualized by autoradiography. Specificity of the induced complexes was determined by competition with a 100-fold molar excess of unlabeled AP-1, NF-B, or Sp1 oligonucleotide. Arrows indicate migration of the induced DNA binding complexes. NIC indicates the migration of the non-induced nonspecific gel shift complexes. The migration of the free probe is not shown.

View larger version (47K): [in this window] [in a new window]   Fig. 4.   H2O2 and TNF induce different patterns of AP-1 and NF-B binding activity on the IL-8 promoter in HMEC-1. Nuclear protein extracts from HMEC-1 cells treated with H2O2 (100 µM) or TNF (100 units/ml) for 15, 30, and 60 min were incubated with 32P-end-labeled oligonucleotides from the AP-1-binding site in the IL-8 promoter (A) or the NF-B-binding site in the IL-8 promoter (B). Gel shift complexes were resolved by electrophoresis and visualized by autoradiography. Specificity of the induced complexes was determined by competition with a 100-fold molar excess of unlabeled AP-1, NF-B, or Sp1 oligonucleotide. Arrows indicate migration of the induced DNA binding complexes. NIC indicates the migration of the non-induced nonspecific gel shift complexes. The migration of the free probe is not shown.

TNF Induce Distinct NF-B Binding Complexes in HMEC-1 and A549-- To demonstrate that the IL-8 and consensus oligonucleotides were indeed binding similar AP-1 and NF-B proteins, we performed a series of competition experiments. As shown in Fig. 5, the AP-1 and NF-B binding complexes formed on the IL-8 promoter sites were competed by a 100-fold molar excess of the appropriate consensus oligonucleotide, indicating the IL-8 promoter sites also recognize and bind consensus AP-1 and NF-B binding complexes. Although the consensus NF-B-binding sites competed with the IL-8 site, a comparison of the NF-B binding complexes in A549 and HMEC-1 revealed differences in mobility of the induced complexes (Fig. 5B). In A549 cells, the predominant NF-B binding activity migrated as a single complex between the non-induced, nonspecific complexes (NIC), whereas in HMEC-1 the predominant NF-B binding activity migrated as a cluster of complexes above the non-induced complex. Because NF-B is a family of proteins that bind as heterodimers, it is not unusual to detect multiple NF-B specific complexes. These data indicate that distinct NF-B binding complexes are selectively induced by TNF in A549 and HMEC-1. In contrast to NF-B, the AP-1 binding complexes induced by H₂O₂ or TNF comigrated in the two cell types (Fig. 5A), suggesting similar AP-1 binding complexes are induced by H₂O₂ and TNF.

View larger version (49K): [in this window] [in a new window]   Fig. 5.   TNF induce distinct NF-B binding complexes in HMEC-1 and A549. Nuclear protein extracts from A549 and HMEC-1 cells treated with H2O2 (800 and 100 µM, respectively) or TNF (100 units/ml) for 60 min were incubated with 32P-end-labeled oligonucleotides from the AP-1-binding site in the IL-8 promoter (A) or the NF-B-binding site in the IL-8 promoter (B). Gel shift complexes were resolved by electrophoresis and visualized by autoradiography. Specificity of the induced complexes was determined by competition with a 100-fold molar excess of unlabeled consensus cAP-1 or consensus cNF-B oligonucleotide. Arrows indicate migration of the induced DNA binding complexes. NIC indicates the migration of the non-induced nonspecific gel shift complexes. The migration of the free probe is not shown.

Mutation of the IL-8 Promoter Binding Sites Abrogate NF-B and AP-1 Binding Activity-- To determine if mutations of the AP-1 and NF-B-binding sites in the IL-8 promoter alter AP-1 and NF-B binding activity in A549 and HMEC-1, we introduced point mutations in the IL-8 promoter oligonucleotides (Table I) that were previously reported to reduce IL-8 promoter activity (18, 32). As shown in Fig. 6A, mutation of the AP-1 or NF-B-binding sites disabled their ability to compete with their respective wild type binding sites. In addition, as shown in Fig. 6, B and C, no induced DNA binding activity was detected on the mutant oligonucleotide probes. These data demonstrate that mutation of the AP-1 and NF-B-binding sites in the IL-8 promoter that affect function also prevent H₂O₂ and TNF induced DNA binding activity.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Mutation of the IL-8 promoter binding sites abrogate NF-B and AP-1 binding activity. Nuclear protein extracts from A549 cells treated with H2O2 (800 µM) or TNF (100 units/ml) for 60 min were incubated with 32P-end-labeled oligonucleotides from the AP-1 or NF-B-binding site in the IL-8 promoter (A) or oligonucleotides containing mutations in the AP-1 (B) or NF-B-binding sites (C) as indicated in Table I. Gel shift complexes were resolved by electrophoresis and visualized by autoradiography. Specificity of the induced complexes was determined by competition with a 100-fold molar excess of unlabeled AP-1, AP-1m, NF-B, or NF-Bm oligonucleotide. Arrows indicate migration of the induced DNA binding complexes. NIC indicates the migration of the non-induced nonspecific gel shift complexes. The migration of the free probe is not shown.

Composition of the AP-1 and NF-B Binding Complexes Induced by H₂O₂ and TNF-- To identify the AP-1 and NF-B components induced in A549 and HMEC-1, we used antibodies to AP-1 and NF-B subunits in supershift assays. As shown in Fig. 7, TNF and H₂O₂ induced the same AP-1 components in A549 cells. Antibodies to c-Fos and JunD produced supershifts of the AP-1 complex induced by H₂O₂ or TNF (Fig. 7A). In contrast, antibodies to c-Jun produced no supershifted complexes relative to the control IgG antibody. These data indicate that H₂O₂ and TNF induce c-Fos/JunD heterodimers in A549 cells.

View larger version (49K): [in this window] [in a new window]   Fig. 7.   Composition of the AP-1 binding complexes induced by H2O2 and TNF. Nuclear protein extracts from A549 and HMEC-1 cells treated with H2O2 (800 and 100 µM, respectively) or TNF (100 units/ml) for 60 min were incubated with 32P-end-labeled oligonucleotides from the AP-1-binding site in the IL-8 promoter (A) or the NF-B-binding site in the IL-8 promoter (B). Antibodies against AP-1 components c-Jun, c-Fos, and JunD, and antibodies against NF-B components Rel A (p65), p50, and cRel were added to the binding reaction as indicated above each lane. Gel supershift complexes were resolved by electrophoresis and visualized by autoradiography. SS indicates the migration of the antibody supershift complexes. NIC indicates the migration of the non-induced nonspecific gel shift complexes. The migration of the free probe is not shown. Note that antibodies to JunD, c-Fosl, and Rel A (p65) generated supershift complexes.

Although TNF induced multiple gel shift complexes, we were only able to detect Rel A (p65) as a component of the NF-B binding complexes in A549 and HMEC-1. As shown in Fig. 7B, Rel A (p65) antibodies supershifted NF-B complexes induced by TNF in both cell types. In contrast, no supershifts were detected with p50 or cRel antibodies. In control experiments, the anti-p50 antibody did supershift NF-B binding complexes derived from monocytic cells, indicating that the p50 antibody was functional (data not shown).

To directly demonstrate that H₂O₂ did not activate Rel A in HMEC-1 and A549 cells, we examined nuclear translocation of Rel A by immunohistochemistry. In resting cells, NF-B resides primarily in the cytoplasm complexed with an inhibitor protein I-B (33). Cellular activation leads to the phosphorylation of I-B, which target the inhibitor protein for ubiquination and then proteolysis by the proteasome (34). Proteolytic degradation of I-B permits NF-B to translocate to the nucleus and bind its recognition site in the promoter of IL-8 (35). To examine NF-B activation and nuclear translocation, HMEC-1 and A549 cells were treated with H₂O₂ or TNF for 60 min and stained with a rabbit anti-Rel A (p65) antibody. Localization of Rel A was detected using a fluorescein-tagged goat anti-rabbit secondary antibody. As shown in Fig. 8, TNF dramatically increased the nuclear staining of the two cell types, whereas little or no increase in nuclear fluorescence was observed in either HMEC-1 or A549 cells stimulated with H₂O₂. These data support the DNA binding studies demonstrating that TNF but not H₂O₂ activates NF-B in HMEC-1 and A549 cells.

View larger version (115K): [in this window] [in a new window]   Fig. 8.   TNF but not H2O2 induces translocation of Rel A. A549 and HMEC-1 were treated with H2O2 or TNF for 1 h and assessed for nuclear translocation of Rel A by immunostaining using an anti-Rel A antibody and a fluorescein isothiocyanate-conjugated secondary antibody. Staining was observed under a fluorescent microscope. Note the prominent nuclear staining of the TNF-treated cells.

H₂O₂ Induction of IL-8 mRNA Expression Correlates with AP-1 Activation-- H₂O₂ induces IL-8 mRNA expression and protein secretion in a dose-dependent fashion in A549 with optimal induction between 400 and 800 µM H₂O₂ (20, 21). To relate AP-1 binding activity to IL-8 gene expression, we compared the induction of AP-1 binding activity on the IL-8 promoter and IL-8 mRNA expression in response to increasing concentrations of H₂O₂. As shown in Fig. 9, H₂O₂ increased IL-8 mRNA expression and AP-1 binding activity in a concentration-dependent manner. The concentration-dependent increase in IL-8 mRNA expression coincided with the concentration-dependent increase in AP-1 binding activity. These data suggest that H₂O₂ induction of IL-8 gene expression in A549 cells is mediated by AP-1 binding to the IL-8 promoter.

View larger version (89K): [in this window] [in a new window]   Fig. 9.   IL-8 expression correlates with H2O2 activation of AP-1. Total RNA or nuclear protein was isolated from A549 cells treated with increasing concentrations of H2O2 for 3 h or 1 h, respectfully. Induction of IL-8 mRNA expression was assessed by Northern blot (lower panel) and induction of AP-1 binding activity was assessed by EMSA using the AP-1 oligonucleotide from the IL-8 promoter (upper panel). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene is shown as an internal control for RNA loading. Note that the dose responses for IL-8 mRNA expression and AP-1 binding activity are essentially identical.

Thiol Oxidation Inhibits H₂O₂ Induction of AP-1 Activity and IL-8 Expression-- AP-1 and NF-B binding activity is mediated by a conserved redox-sensitive cysteine residue within the DNA-binding domain (36, 37). Thiol oxidation agents such as diamide can inhibit AP-1 and NF-B binding activity (38). To determine whether thiol oxidation of AP-1 or NF-B could modulate IL-8 gene expression, we examined the effect of diamide on H₂O₂ induction of AP-1 activity and IL-8 protein secretion. Nuclear protein extracts from A549 cells pretreated for 60 min with increasing concentrations of diamide followed by stimulation with 800 µM H₂O₂ for 60 min were assessed for AP-1 binding activity. As shown in Fig. 10, diamide dose dependently inhibited H₂O₂ induced AP-1 binding activity to the IL-8 promoter with complete inhibition at 800 µM diamide. These data demonstrate that diamide-mediated thiol oxidation inhibits H₂O₂ induction of AP-1 binding to the IL-8 promoter in A549 cells.

View larger version (76K): [in this window] [in a new window]   Fig. 10.   Effect of diamide on H2O2-induced AP-1 binding activity in A549 cells. Cells were pretreated with increasing concentrations of diamide for 60 min and then stimulated with H2O2 (800 µM) as indicated above each lane. Nuclear protein extracts were prepared 60 min post-treatment and assessed for AP-1 binding activity by EMSA using the AP-1 oligonucleotide corresponding to the AP-1-binding site in the IL-8 promoter. NIC indicates the migration of the non-induced nonspecific gel shift complexes. The migration of the free probe is not shown.

To relate the diamide effect on AP-1 binding activity with IL-8 expression, we measured culture supernatants at 24 h for IL-8 protein secretion. As shown in Fig. 11, diamide dose dependently inhibited the H₂O₂ induction of IL-8 protein secretion, consistent with the close association between AP-1 binding activity and IL-8 gene expression in A549 cells. However, in contrast to the effect of diamide on AP-1 binding activity, IL-8 secretion was inhibited by much lower concentrations of diamide, suggesting that thiol oxidation can also effect IL-8 expression independently of AP-1 binding activity. Moreover, the H₂O₂ induced IL-8 secretion was more sensitive to diamide than the spontaneous secretion of IL-8, suggesting that the constitutive and induced mechanisms of IL-8 expression may differ in A549 epithelial cells. Taken together, these data indicate that H₂O₂ induction of IL-8 in A549 cells is mediated by complex redox mechanisms involving AP-1 binding to the IL-8 promoter.

View larger version (17K): [in this window] [in a new window]   Fig. 11.   Effect of diamide on H2O2-induced IL-8 secretion in A549 cells. Cells were pretreated with increasing concentrations of diamide for 60 min and then stimulated with H2O2 (800 µM) as indicated below the histogram. Supernatants were collected 24 h post-treatment and assessed for IL-8 protein content by enzyme-linked immunosorbent assay. The mean of three independent experiments plus the S.E. are shown.

	DISCUSSION

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IL-8 and ICAM-1 are critical protein factors in the recruitment of leukocytes to sites of inflammation and oxidant stress is an important regulator of their expression (13, 20). The up-regulation of ICAM-1 on the surface of endothelium is required for the firm adhesion of rolling neutrophils and a chemotactic gradient of IL-8 is critical for the adherent neutrophils to migrate across the alveolar-capillary membrane during lung inflammation and injury (39). In this study, we investigated the mechanism by which IL-8 and ICAM-1 are differentially regulated by oxidant stress in epithelial and endothelial cells. As summarized in Table II, we demonstrate that H₂O₂ differentially activates the redox-responsive transcription factors AP-1 and NF-B in epithelial and endothelial cells. H₂O₂ selectively induced AP-1 in A549 cells, whereas TNF induced both AP-1 and NF-B (Table II). Moreover, H₂O₂ induction of AP-1 binding to the IL-8 promoter was closely associated with H₂O₂ induction of IL-8 mRNA expression, suggesting that H₂O₂ induces IL-8 in epithelial cells through the action of the transcription factor AP-1. In contrast, in HMEC-1, AP-1 binding activity on the IL-8 promoter was constitutive and H₂O₂ did not stimulate an increase with this basal AP-1 binding activity. H₂O₂ also did not stimulate NF-B binding activity in HMEC-1, whereas TNF increased only NF-B binding activity (Table II). Consistent with the DNA binding studies, we recently showed that TNF but not H₂O₂ could induce IL-8 promoter activity in HMEC-1.²

View this table: [in this window] [in a new window]   Table II Summary of AP-1 and NF-B binding activities induced by H2O2 and TNF in HMEC-1 and A549 cells These data represent a summary of the gel shift data presented in Figs. 1-5. Plus signs indicate that induced binding activity was detected. Minus signs indicate that little or no induced binding activity was detected over untreated control cells. Note the different patterns of AP-1 and NF-B binding activity induced in the two cell types. TF, transcription factor.

In contrast to H₂O₂, TNF appears to induce IL-8 in epithelial and endothelial cells mostly through the activation of NF-B. As with AP-1, a redox mechanism appears to be involved since diamide, which is known to target a conserved cysteine residue in the DNA-binding domain of NF-B, inhibited TNF induced IL-8 expression.² This conclusion is consistent with several studies demonstrating that TNF activates IL-8 and ICAM-1 transcription through a cooperative interaction between NF-B and NF-IL-6 (C/EBP-) (17, 19). Indeed, we found little or no binding activity on the IL-8 NF-IL-6 binding site,² consistent with the binding requirement of NF-B for NF-IL-6 to also bind to the IL-8 promoter (17). In agreement with our findings, several studies have shown that H₂O₂ also does not activate NF-B in primary endothelial cells (13, 40, 41). However, H₂O₂ has been reported to activate NF-B in porcine aortic endothelial cells and transformed endothelial cell lines (40, 42, 43), indicating H₂O₂ activation of NF-B is complex and may depend on the endothelial cell type. In contrast to endothelial cells, in A549 epithelial cells, TNF induced, in addition to NF-B, AP-1 binding activity on the IL-8 promoter, suggesting AP-1 may contribute to the TNF induction of IL-8 in epithelial cells. Indeed, the duel binding of AP-1 and NF-B to the IL-8 promoter may account for the greater induction of IL-8 expression by TNF than H₂O₂ in A549 cells.

Oxidant stress has been reported to affect AP-1 and NF-B differently, suggesting distinct mechanisms of redox regulation of these transcription factors (44, 45). Our studies suggest that H₂O₂ and TNF regulate IL-8 and ICAM-1 gene expression through distinct signal transduction pathways, and are consistent with the data of Das et al. demonstrating differential redox regulation of AP-1 and NF-B in A549 cells (46). They found that thiols induced NF-B but not AP-1, while oxidants induced AP-1 but not NF-B. Oxidants such as ozone have also been shown to activate IL-8 expression through AP-1 and NF-B in A549 cells (47). AP-1-like proteins have also been associated with the transcriptional regulation of -glutamylcysteine synthetase-heavy subunit by oxidants in A549 cells (48). The differential activation of AP-1 and NF-B has also been shown in other cell systems (44, 45, 49-53). We conclude that the redox regulation of AP-1 and NF-B contribute to the distinct patterns of gene expression induced in epithelial and endothelial cells by H₂O₂ and TNF. A similar finding has been reported for the manganese superoxide dismutase gene in pulmonary epithelial cells (54). However, in contrast to A549 cells, the H441 pulmonary epithelial cell line did not mediate H₂O₂ or TNF induction of AP-1, suggesting H₂O₂ and TNF induce manganese superoxide dismutase expression independent of AP-1 activity. Thus, H₂O₂ and TNF can activate gene expression in epithelial cells via both AP-1 dependent and independent mechanisms. This is consistent with our diamide studies demonstrating that the thiol oxidation sensitivity of IL-8 expression was considerably greater than that of the AP-1 binding activity.

AP-1 is composed of heterodimers of Jun and Fos proteins and oxidant stress increases the transcription of c-jun and c-fos gene expression (55). Although c-jun has been demonstrated to be activated by H₂O₂ in epithelial cells (56), we did not detect any c-Jun protein in the H₂O₂ induced AP-1 complexes in A549 cells. Instead, another member of the Jun family, JunD, was induced by H₂O₂. H₂O₂ also rapidly activates c-fos gene expression (57-60), and indeed we detected increased c-Fos protein within 1 h in AP-1 binding complexes induced by H₂O₂. The data indicate that H₂O₂ induces c-Fos/JunD heterodimers and JunD/JunD homodimers in A549 cells. With regard to NF-B, we were able to detect only the Rel A (p65) subunit in the nucleus of HMEC-1 and A549 cells, consistent with the findings of Kunsch and Rosen (61) demonstrating Rel A binding to the IL-8 promoter in Jurkat T-cells. Although H₂O₂ does not induce NF-B in A549 cells, other modes of oxidant stress may activate NF-B. For example, oxidant stress induced by pyrogallol, a superoxide generator, can increase NF-B binding activity in A549 cells (62).

The redox regulation of AP-1 and NF-B may also be involved in the differential expression of CC chemokines. We reported previously that H₂O₂ induces MCP-1 but not RANTES in A549 cells, whereas TNF induced both chemokines (20). MCP-1 has been reported to be regulated by redox mechanisms (63-65), involving NF-B and AP-1 (66-70), and RANTES has recently been shown to be regulated by NF-B (71). We propose that the differential activation of redox-responsive transcription factors like AP-1 and NF-B set up distinct patterns of gene expression in epithelial and endothelial cells that may critically influence the site-specific recruitment of leukocyte subsets during inflammatory responses.

The differential activation of AP-1 and NF-B by H₂O₂ and TNF are consistent with our previous results demonstrating that H₂O₂ and TNF activate ICAM-1 gene expression through distinct cis-acting elements in the ICAM-1 promoter (13). H₂O₂ activated ICAM-1 transcription through an element of the promoter that contain antioxidant responsive elements (ARE), whereas TNF targeted the proximal promoter containing a composite binding site for NF-B and NF-IL-6 (13). It was previously demonstrated that TNF activates ICAM-1 and IL-8 through the cooperative interaction of the NF-B and NF-IL-6-binding sites (7, 17-19). H₂O₂ induces ICAM-1 expression in HMEC-1 (20), even though H₂O₂ does not induce AP-1 or NF-B in these endothelial cells. In contrast, H₂O₂ does not induce ICAM-1 in A549 cells even though AP-1 is activated. These data suggest that H₂O₂ induction of ICAM-1 in endothelial cells is not mediated by AP-1 or NF-B, but more likely by a novel redox-responsive transcription factor.

In EAhy926, an epithelial/endothelial hybrid cell line generated from the fusion of A549 and human umbilical vein endothelial cells, H₂O₂ induces both ICAM-1 and IL-8, indicating that fusion of endothelial and epithelial cells abrogates their ability to differentially regulate the two genes (13, 20). The dominance of the H₂O₂ gene induction suggests the involvement of positive trans-acting factors in the discordant oxidant regulation of ICAM-1 and IL-8 in epithelial and endothelial cells. ICAM-1 expression was induced through a H₂O₂ responsive region of the promoter containing tandem 16-base pair AP-1/Ets composite sites (13). These AP-1/Ets repeats also have homology to known antioxidant responsive elements (ARE) first identified as redox-responsive elements in GST Ya subunit gene (72-76). AP-1 can physically interact with Ets at AP-1/Ets composite sites to functionally activate gene transcription in response to H₂O₂ (77-79). H₂O₂ increased binding activity on the ICAM-1 ARE sites and mutation of either the AP-1 or Ets motif abrogated the H₂O₂ induced binding activity (13). However, in A549 cells, ICAM-1 expression is not stimulated by H₂O₂, whereas H₂O₂ induced ICAM-1 in HMEC-1 (20). Since H₂O₂ did not induce AP-1 or NF-B in HMEC-1, it appears that H₂O₂ induction of ICAM-1 in endothelial cells is mediated by novel redox responsive transcription factors. ARE-binding proteins capable of binding the ICAM-1 AP-1/Ets sites have recently been identified providing a potential mechanism by which H₂O₂ could activate ICAM-1 in endothelial cells independently of AP-1 or NF-B (80-82). Indeed, oxidant stress can differentially induce other transcription factors in epithelial and endothelial cells. For example, we previously demonstrated that H₂O₂ differentially induces the winged-helix transcription factor, HFH-11 in A549 and HMEC-1 (83). H₂O₂ increased HFH-11 expression in HMEC-1 but not in A549 cells (20).

In conclusion, we have demonstrated that H₂O₂ and TNF can differentially activate the redox-responsive transcription factors AP-1 and NF-B in epithelial and endothelial cells. We propose this differential regulation of AP-1 and NF-B leads to different patterns of gene expression in epithelial and endothelial cells that may be critical for their function during oxidant stress.

	ACKNOWLEDGEMENTS

We thank M. Grover for technical assistance with immunostaining and Dr. D. Rubin for assistance in obtaining micrographs of immunostained cells.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant AR45835 and by a Grant-In-Aid from the American Heart Association (to K. A. R.), and grants from the American Lung Association (Chicago) and the American Cancer Society of Illinois (to K. A. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present Address: Dept. of Medicine, The Section of Cardiovascular Sciences, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.

^§ Present Address: Dept. of Medicine, University of Chicago, Chicago, IL 60637.

^¶ To whom correspondence and reprint requests should be addressed: Dept. of Immunology/Microbiology, Rush Medical College, 1653 W. Congress Pkwy., Chicago, IL 60612. Tel.: 312-942-6259; Fax: 312-942-2808; E-mail:kroebuck{at}rush.edu.

The abbreviations used are: IL, interleukin; TNF, tumor necrosis factor-; NF-B, nuclear factor-B; PBS, phosphate-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; HMEC, human microvessel endothelial cell; EMSA, electrophoretic mobility shift assay; NIC, nonspecific complexes; ICAM-1, intercellular adhesion molecule (CD54); ARE, antioxidant responsive elements.

² V. Lakshminarayanan and K. A. Roebuck, unpublished data.

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