E-selectin expression in human endothelial cells by TNF-alpha -induced oxidant generation and NF-kappa B activation

Arshad Rahman, John Kefer, Masashi Bando, Walter D. Niles, and Asrar B. Malik

Department of Pharmacology, College of Medicine, The University of Illinois, Chicago, Illinois 60612

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Because reactive oxygen species (ROS) can function as second messengers and regulate the activation of the transcription factor nuclear factor (NF)-kappa B, we investigated the possible role of tumor necrosis factor-alpha (TNF-alpha )-induced ROS generation in endothelial cells in signaling E-selectin gene transcription. We demonstrated that stimulation of human pulmonary artery endothelial cells with TNF-alpha (100 U/ml) resulted in ROS production using the oxidant-sensitive dye 5 (and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate bis(acetoxymethyl)ester. Pretreatment with N-acetyl-L-cysteine (NAC) or pyrrolidine dithiocarbamate (PDTC) for 0.5 h inhibited TNF-alpha -induced generation of ROS as well as activation of NF-kappa B and E-selectin mRNA and the cell surface protein expression. These findings indicate that TNF-alpha induces NF-kappa B activation and the resultant E-selectin gene expression by a pathway that involves formation of ROS and that E-selectin expression can be inhibited by the antioxidant action of NAC or PDTC. The results support the hypothesis that generation of ROS in endothelial cells induced by proinflammatory cytokines such as TNF-alpha is a critical signal mediating E-selectin expression.

endothelium; tumor necrosis factor-alpha ; reactive oxygen species; nuclear factor-kappa B

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

E-SELECTIN (CD62E), a member of the selectin family of cell surface glycoproteins, is an endothelial cell-specific adhesion protein involved in the initial vessel wall binding and rolling of polymorphonuclear leukocytes (PMN) and other leukocytes at sites of endothelial activation (34). E-selectin ligands on PMN are fucose-rich glycoproteins containing sialylated Lewisx and related oligosaccharides (14, 20). E-selectin is expressed on the endothelial cell surface in response to cytokines such as tumor necrosis factor-alpha (TNF-alpha ) and interleukin-1beta (IL-1beta ; see Refs. 6 and 7). Transcription of the E-selectin gene is tightly regulated in a cell-specific manner because it is expressed exclusively on the endothelial cell surface and its expression is dependent in major part on activation of the transcription factor nuclear factor (NF)-kappa B after exposure to TNF-alpha (11, 21, 27, 35, 38, 45). NF-kappa B, a heterodimer of 50-kDa (p50) and 65-kDa (p65) subunits, exists in the cytoplasm of most cells, including endothelial cells in an inactive form bound to the inhibitory protein I-kappa B (Ikappa B) through the p65 molecule (3, 5). Treatment of cells with TNF-alpha leads to activation of Ikappa B kinases alpha  and beta (IKKalpha and IKKbeta ; see Refs. 12 and 46), which phosphorylate Ikappa Balpha and Ikappa Bbeta , respectively, at two conserved NH2-terminal residues and which in turn targets them for rapid polyubiquitination followed by degradation through the 26S proteasome (8-10, 43). This results in release of NF-kappa B dimer and its translocation to the nucleus to activate transcription of genes involved in immune and inflammatory responses (2-4). Genetic analysis has identified three NF-kappa B sites in the human E-selectin gene that are essential for maximal promoter activity after cytokine exposure (38): NF-kappa B sites 1 and 2 (5'-CGTGGATATTCCCGGGAAAGTTTTT-3', element underlined, within -129 to -105 bases from the transcriptional start site) and NF-kappa B site 3 (5'-CATT<UNL>GGGGATTTCC</UNL>TCTTT-3', element underlined, within -99 to -80 bases from the transcriptional start site). Studies have shown that NF-kappa B binds preferentially to sites 2 and 3 and that mutations within these sites are more specific in inhibiting E-selectin expression than site 1 mutations (38).

TNF-alpha stimulates the production of reactive oxygen species (ROS): superoxide anion, hydrogen peroxide, and hydroxyl radicals in a variety of cell types (19, 23, 32). ROS generated in excess amounts damages cells by peroxidizing lipids and disrupting proteins and nucleic acids. ROS in lower concentrations may function as second messengers in mediating TNF-alpha - and IL-1beta -activated signal transduction pathways that regulate the transcription factors NF-kappa B and activator protein (AP)-1 (22, 33). However, there are conflicting reports regarding oxidant-induced expression of E-selectin through NF-kappa B activation (16). Studies have shown that the antioxidant pyrrolidine dithiocarbamate (PDTC) inhibited cytokine-induced vascular cell adhesion molecule-1 (VCAM-1) expression by preventing NF-kappa B activation but surprisingly failed to prevent E-selectin expression (24, 44). Because p65 alone or in concert with the p50 subunit of NF-kappa B is essential for regulation of these adhesion molecules in cytokine-activated endothelial cells (11, 38, 41, 45), it is expected that there would be a common redox-sensitive mechanism controlling the expression of both VCAM-1 and E-selectin. The differential sensitivity of E-selectin and VCAM-1 expression to antioxidants may be ascribed to factors such as 1) specific arrangement and number of NF-kappa B sites present in promoters of E-selectin versus VCAM-1 (17, 38), 2) codependency of NF-kappa B on other transcription factors in E-selectin gene (21, 31, 36), 3) nature of the antioxidant, and 4) differences in DNA binding sites in the promoter of these genes for antioxidant-responsive transcription factors. In view of the conflicting evidence on the signaling function of oxidants in inducing E-selectin expression, in the present study, we have reevaluated the role of TNF-alpha in stimulating ROS production in endothelial cells and in regulating NF-kappa B activation and the E-selectin mRNA and protein expression.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell culture. Human pulmonary artery endothelial (HPAE) cells at the first passage were obtained from Clonetics and were grown on gelatin-coated flasks or plates in endothelial cell growth medium (EGM) containing 10% fetal calf serum (FCS) and 3.0 mg/ml of endothelial-derived growth factor from bovine brain extract protein. Human recombinant TNF-alpha with a specific activity of 2.3 × 107 was purchased from Promega (Madison, WI). All experiments were performed on cells under the 10th passage except where otherwise indicated. Confluent HPAE cells were washed two times with serum-free DMEM (without phenol red) containing 20 mM HEPES or EGM containing 2% FCS and then were incubated in the same medium with the indicated concentrations of N-acetyl-L-cysteine (NAC) or PDTC for 0.5 h before TNF-alpha was added. TNF-alpha was added directly to the medium containing NAC or PDTC for the times and at the concentrations indicated in each experiment. NAC was neutralized to pH 7.0 before use in all experiments.

Northern blot analysis. Total RNA was isolated from HPAE cells with TRIzol (GIBCO BRL) according to manufacturer's recommendations. Quantification and purity of RNA were assessed by the ratio of absorbance at 260 nm to that at 280 nm, and an aliquot of RNA (20 µg) from samples with a ratio above 1.6 was fractionated using a 1% agarose-formaldehyde gel. The RNA was transferred to a Duralose-ultraviolet (UV) nitrocellulose membrane (Stratagene, La Jolla, CA) and covalently linked by UV irradiation using a Stratalinker UV cross-linker (Stratagene). Human E-selectin (1.35-kb EcoR I fragment; see Ref. 7) and rat glyceraldehyde-3-phosphate dehydrogenase (1.1-kb Pst I fragment) were labeled with [alpha -32P]dCTP using the random-primer kit (Stratagene), and hybridization was carried out as described (37). Briefly, the blots were prehybridized for 30 min at 68°C in QuikHyb solution (Stratagene) and hybridized for 2 h at 68°C with random-primed 32P-labeled probes. After hybridization, the blots were washed two times for 30 min at room temperature in 2× saline-sodium citrate (SSC) with 0.1% SDS followed by two washes for 30 min each at 60°C in 0.1× SSC with 0.1% SDS. Autoradiography was performed with an intensifying screen at -70°C for 12-24 h. The signal intensities were quantified by scanning the autoradiograms with a laser densitometer (Howtek, Hudson, NH) linked to a computer analysis system (PDI, Huntington Station, NY). The nitrocellulose membrane was soaked for stripping the probe with boiled water or 0.1× SSC with 0.1% SDS.

Detection of ROS generation with C-DCHF-DA staining. Confluent HPAE monolayers were pretreated for 0.5 h with NAC (30 mM) or PDTC (100 µM) and stimulated for 1 h with TNF-alpha (100 U/ml) in EGM containing 2% serum. After treatment, cells were washed two times with EGM (with 2% serum) and stained for 20 min with 2.5 µM 5 (and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate bis(acetoxymethyl)ester (C-DCHF-DA; Molecular Probes, Eugene, OR) in EGM (with 2% serum) as described (15). Cells were viewed with fluorescence microscopy and photographed. Quantitative fluorescence images were obtained on a Nikon Diaphot 200 Microscope (Nikon, Garden City, NJ) using Image Pro Plus software (Media Cybernetics, Silver Spring, MD). Each sample was independently stained so that the samples were exposed to the dye for the same time. The dye solution was freshly prepared in prewarmed EGM (with 2% serum) for each sample. After being stained for 20 min at 37°C, samples were rinsed two times with EGM (with 2% serum) containing no dye and were scanned on a fluorescence microscope. Samples were epi-illuminated by a 150-watt Hg lamp and viewed with fluorescein filters (B2E cube). Fields were observed at ×20 numerical aperture 0.4 and were acquired with a charge-coupled device imaging array (Photometrics, Tucson, AZ) under computer control with 1-s integration time. Illumination caused increased fluorescence because of oxidation of the dye; therefore, each field was exposed to light for exactly the same time. The image size for scanning was 768 horizontal × 468 vertical. The average relative fluorescence intensity for every cell in each field was determined using Imag Proplus software (Media Cybernetics).

Nuclear extract preparation. HPAE cells were pretreated for 0.5 h without or with NAC (30 mM) and then were left untreated or stimulated for 1 h with TNF-alpha (100 U/ml). Cells were washed two times with ice-cold Tris-buffered saline and resuspended in 400 µl of 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). After 15 min, Nonidet P-40 was added to a final concentration of 0.6%. Nuclei were pelleted and resuspended in 50 µl of 20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF. After 30 min at 4°C, the lysates were centrifuged, and supernatants containing the nuclear proteins were transferred to new vials. The protein concentration of the extract was measured using a Bio-Rad protein determination kit (Bio-Rad, Hercules, CA).

Electrophoretic mobility shift assay. Electrophoretic mobility shift assays were performed by incubating 10 µg of nuclear extract with 1 µg of poly(dI-dC) in a binding buffer [10 mM Tris · HCl (pH 7.5), 50 mM NaCl, 0.5 mM DTT, and 10% glycerol (20 µl final volume)] for 15 min at room temperature. Next, end-labeled double-stranded oligonucleotides containing the NF-kappa B binding site of E-selectin promoter (30,000 counts/min each) were added in the absence or presence of 25- or 100-fold molar excess of cold competitor, and the reaction mixtures were incubated for 15 min at room temperature. The DNA-protein complexes were resolved in 5% native polyacrylamide gel electrophoresis in low-ionic-strength buffer (0.25× Tris-borate-EDTA). Oligonucleotides used for the gel shift analysis were as follows: E-selectin NF-kappa BA, 5'-TTTTAAGCATCGT<UNL>GGATATTCCC</UNL><UNL>GGGAAAG</UNL>TTTTT-3' (underlined sequences represent sites 1 and 2, respectively); mut-E-selectin NF-kappa BA, 5'-TTTTAAGCATCGTGGATATTggCGGccgccTTTTT-3'; E-selectin NF-kappa BB, 5'-TTTTTGGATGCCATT<OVL>GGGGATTTCC</OVL>TCTTT-3' (underlined sequence represents site 3); mut-E-selectin NF-kappa BB, 5'-TTTTTGGATGggcggccGccTTTCCTCTTT-3'; Igkappa B, 5'-AGTTGAGGGGACTTTCCCAGGC-3'; Oct-1 DNA, 5'-AATTGCATGCCTGCAGGTCGACTCTAGAGGATCC<UNL>ATGCAAAT</UNL>GCCGGGTACCGAGCTC-3'.

E-selectin promoter is characterized by the presence of three NF-kappa B sites located 126 (site 1), 116 (site 2), and 94 (site 3) bp upstream relative to the transcription start site (38). The oligonucleotides E-selectin NF-kappa BA and E-selectin NF-kappa BB represent the 35 (-139 to -105)- and 30 (-110 to -81)-bp sequences, respectively, of E-selectin promoter. The oligonucleotide E-selectin NF-kappa BA encompasses NF-kappa B site 1 (-126) and NF-kappa B site 2 (-116), whereas the oligonucleotide E-selectin NF-kappa BB contains the NF-kappa B site (-94) of the promoter. The oligonucleotides mut-E-selectin NF-kappa BA and mut-E-selectin NF-kappa BB represent the NF-kappa B-mutated versions of E-selectin NF-kappa BA and E-selectin NF-kappa BB oligonucleotides, respectively. Similarly, Igkappa B oligonucleotide contains the consensus NF-kappa B binding site present in the immunoglobulin gene. The oligonucleotide Oct-1, which contains a binding site for Oct-1, was used to serve as a negative control in competition experiments. Sequence motifs within the oligonucleotides are underlined, and the mutations are shown in lowercase letters.

Reporter gene constructs, endothelial cell transfection, and luciferase assay. The E-selectin luciferase (Luc) plasmid containing ~782 bp of E-selectin 5'-flanking DNA, linked to the firefly Luc reporter gene, has been described previously (38). Cells under the fifth passage were plated into six-well Primaria culture dishes 18-24 h before transfection. Transfections were performed with lipofectine (GIBCO BRL) as described (37). Briefly, reporter DNA (1 µg) was mixed with 2 µl of lipofectine in 200 µl of Opti-MEM I (GIBCO BRL). We used the plasmid pSVgal (0.2 µg) containing the beta -galactosidase gene driven by the constitutively active SV40 promoter to normalize the transfection efficiencies. Because we did not observe any significant difference in transfection efficiencies in the initial experiments, we did not cotransfect the pSVgal construct in the later experiments. After a 30-min incubation, Opti-MEM I (800 µl) was added, and the mixture was applied on the cells that had been washed two times with Opti-MEM I. At 3 h later, the medium was changed to EGM containing 10% serum, and the cells were harvested 24-48 h after transfection. Using this protocol, we achieved a transient transfection efficiency of 15 ± 3% (mean ± SD; n = 3 experiments) for HPAE cells. Recombinant TNF-alpha was used at a concentration of 100 U/ml for 12-18 h before the cells were harvested. Cell extract was prepared and assayed for Luc activity using the Promega Biotech assay system (Promega) and beta -galactosidase activity using the Tropix assay system (Bedford, MA). Luc activity per microgram of protein extract was normalized to beta -galactosidase activity. The protein content was determined using a Bio-Rad protein determination kit.

Flow cytometry analysis. HPAE cells cultured in six-well Primaria tissue culture dishes were pretreated for 0.5 h with 30 mM NAC or 100 µM PDTC and then 100 U/ml TNF-alpha were added without removing the antioxidant. After completion of the incubation period, cells were washed two times with cold PBS, removed by careful trypsinization, incubated in 20% FCS for 30 min at 4°C, and washed two times with cold PBS. Cells were incubated at 4°C for 30 min with monoclonal antibody against E-selectin (BBA1 obtained from R&D Systems, Minneapolis, MN). Samples were analyzed by flow cytometry in a FACScan cytofluorometer (Becton Dickinson, Mountain View, CA).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

TNF-alpha induces ROS generation in HPAE cells. We used the oxidant-sensitive dye C-DCDHF-DA to detect ROS production in TNF-alpha -stimulated HPAE cells. After uptake, intracellular esterase hydrolyzes the ester bonds, releasing the intact nonfluorescent substrate. This reduced substrate is oxidized by ROS to the fluorescent species carboxydichlorofluorescein, which is retained by living cells. Confluent HPAE monolayers were stimulated with TNF-alpha (100 U/ml) for 1 h and then loaded for 20 min with 2.5 µM C-DCDHF-DA in EGM (with 2% serum) containing no TNF-alpha . Cells were examined for fluorescence intensity by viewing under a fluorescence microscope. Figures 1 and 2 show the fluorescence images of HPAE cells after C-DCDHF-DA staining. Control cells showed a low intensity of fluorescence. In contrast, cells treated with TNF-alpha showed an increased level of fluorescence (the average fluorescence intensity ranged from 5- to 6-fold of control). Preincubation of cells for 0.5 h with NAC (30 mM) or PDTC (100 µM) reduced the TNF-alpha -induced average fluorescence intensity to 1.9- and 2.7-fold, respectively, above the control value. Figures 1 and 2 use histograms showing the results of the above experiment in which fluorescence imaging was used to quantitate the relative fluorescence intensity. The results indicate that TNF-alpha treatment of HPAE cells leads to production of ROS (as indicated by a greater percentage of cells having higher fluorescence intensities) and that this was prevented by preincubation of cells with NAC or PDTC.


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Fig. 1.   N-acetyl-L-cysteine (NAC) inhibits tumor necrosis factor-alpha (TNF-alpha )-induced reactive oxygen species (ROS) production. Confluent human pulmonary artery endothelial (HPAE) cells were pretreated with 30 mM NAC for 0.5 h and then stimulated with TNF-alpha (100 U/ml) for 1 h. Cultures were washed and stained with 2.5 µM 5 (and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate bis(acetoxymethyl)ester (C-DCDHF-DA) dye for 20 min and analyzed by fluorescent microscopy as described in MATERIALS AND METHODS. A: fluorescent images of representative control, TNF-alpha -treated, and NAC-treated HPAE cells before TNF-alpha stimulation (representative of 5 separate experiments). B: relative fluorescence intensities for each condition in A were determined, compiled, and partitioned into 4 brightness classes (1-4), with class 1 representing the lowest fluorescence intensity and class 4 representing the highest fluorescence intensity. Relative fluorescence intensity for cells stimulated with TNF-alpha was markedly shifted to the higher fluorescence intensity classes compared with control cells. Pretreatment with NAC prevented the TNF-alpha -induced shift to the higher fluorescence intensity classes.


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Fig. 2.   Pyrrolidine dithiocarbamate (PDTC) inhibits TNF-alpha -induced ROS production. Confluent HPAE cells were pretreated with 100 µM PDTC for 0.5 h and then were stimulated with TNF-alpha (100 U/ml) for 1 h. Cultures were washed and stained with 2.5 µM CDCDHF-DA dye for 20 min and analyzed by fluorescent microscopy as described in MATERIALS AND METHODS. A: fluorescent images of representative control, TNF-alpha -treated, and PDTC-treated HPAE cells before TNF-alpha stimulation (representative of 3 separate experiments). B: relative fluorescence intensities for each condition in A were determined as described in Fig. 1B.

TNF-alpha -induced ROS generation mediates E-selectin mRNA expression. We used NAC, which readily enters cells and serves as both a scavenger for oxidants (1) and a precursor for glutathione (26), to determine the role of ROS in TNF-alpha -induced E-selectin mRNA expression. Preincubation of HPAE monolayers with NAC (5-30 mM) for 0.5 h caused a dose-dependent decrease in TNF-alpha -induced E-selectin mRNA expression (Fig. 3, A and B), with maximum inhibition of >90% occurring with 30 mM NAC. At a lower concentration of 5 mM, NAC showed no inhibitory effect on TNF-alpha -induced E-selectin mRNA expression, whereas 15 mM NAC prevented ~30% of the TNF-alpha response (Fig. 3, A and B). We determined the effect of NAC on the time course of TNF-alpha -induced E-selectin transcripts. As shown in Fig. 3C, NAC prevented the expression of E-selectin mRNA at all time points tested.


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Fig. 3.   NAC prevents TNF-alpha -induced E-selectin mRNA expression. Confluent HPAE monolayers were pretreated for 0.5 h with 5, 15, and 30 mM (A) or with 30 mM NAC (C), followed by stimulation without or with TNF-alpha (100 U/ml) for a period of 2 h (A) or 0, 0.5, 2, 4, and 8 h (C) in the continuous presence of NAC. Total RNA was isolated and analyzed by Northern hybridizations with a human E-selectin or rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA, which hybridizes to 3.9- or 1.3-kb mRNA transcripts, respectively. GAPDH (a constitutively expressed gene not induced by TNF-alpha ) was used to normalize loading of gels. A and C, autoradiograms; B, bar graph representing the dose-dependent effects of NAC on the relative intensities of E-selectin mRNA signals (representative of 2 separate experiments).

NF-kappa B inhibitor PDTC prevents TNF-alpha -induced E-selectin mRNA expression. We determined the effects of PDTC, which functions as an inhibitor of oxidant-induced NF-kappa B activity through its ability to chelate metal ions and deliver thiol groups to cells (39, 40), on TNF-alpha -induced E-selectin mRNA expression. As shown in Fig. 4, PDTC prevented in a dose-dependent manner E-selectin mRNA expression, suggesting the involvement of ROS in mediating TNF-alpha -induced activation of NF-kappa B and E-selectin gene transcription.


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Fig. 4.   PDTC inhibits TNF-alpha induction of E-selectin transcript. Confluent HPAE cells were preincubated with the indicated concentrations of PDTC for 0.5 h, followed by stimulation with TNF-alpha (100 U/ml) for 1.5 h. E-selectin and GAPDH mRNA expression were determined as described in Fig. 3A. A, autoradiogram; B, bar graph representing the relative intensities of E-selectin mRNA signals (representative of 2 separate experiments).

TNF-alpha -induced ROS generation mediates E-selectin cell surface expression. We evaluated the effects of both NAC and PDTC to determine whether TNF-alpha -induced ROS generation is critical in mediating E-selectin expression on endothelial cell surface. As shown in Fig. 5, exposure of HPAE cells to TNF-alpha for 4 h resulted in a significant increase in E-selectin cell surface expression. Pretreatment for 0.5 h with NAC (Fig. 5A) or PDTC (Fig. 5B) prevented the TNF-alpha -induced cell surface expression of E-selectin.


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Fig. 5.   NAC and PDTC prevent TNF-alpha -induced cell surface expression of E-selectin. Confluent HPAE monolayers were pretreated for 0.5 h with 30 mM NAC (A) or 100 µM PDTC (B) and then stimulated with or without TNF-alpha (100 U/ml) for 4 h. E-selectin expression was assayed by flow cytometry using monoclonal antibody against E-selectin or monoclonal antibody against IgG, as described in MATERIALS AND METHODS. A: comparative fluorescence-activated cell sorter (FACS) profiles of E-selectin expression in untreated (), NAC-treated (bullet ), TNF-alpha -treated (black line), and NAC plus TNF-alpha -treated () cells. B: comparative FACS profiles of E-selectin expression in untreated (), PDTC-treated (bullet ), TNF-alpha -treated (black line), and PDTC plus TNF-alpha -treated () cells. Results are representative of 3 separate experiments.

TNF-alpha -induced ROS generation mediates E-selectin promoter activation. To determine whether TNF-alpha -induced ROS generation mediated E-selectin expression by activating gene transcription, we assessed the effects of NAC on transcriptional activity of the E-selectin promoter after transfection of HPAE cells with an expression plasmid containing firefly Luc gene as a reporter driven by the E-selectin promoter. Treatment with TNF-alpha resulted in an approximately fourfold increase in the activity of E-selectin promoter (Fig. 6A). Pretreatment with NAC prevented TNF-alpha -induced activation of the E-selectin promoter (Fig. 6A). As a control, we transfected HPAE cells with constitutively active cytomegalovirus (CMV) promoter-Luc reporter gene construct (CMV-Luc) and determined the effects of NAC on its transcriptional activity. As shown in Fig. 6B, NAC had no effect on CMV-Luc activity. These results indicate that NAC exerted its inhibitory effects on TNF-alpha -induced E-selectin expression by preventing activation of its promoter.


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Fig. 6.   NAC prevents TNF-alpha -induced activation of E-selectin promoter. HPAE cells were transfected with E-selectin luciferase (Luc) reporter gene construct (A; E-selectin Luc) or cytomegalovirus (CMV) promoter-Luc reporter gene construct (B; CMV-Luc) as described in MATERIALS AND METHODS. Cells were stimulated with TNF-alpha (100 U/ml) without or after pretreatment of NAC (30 mM, 0.5 h) for 12-15 h before cells were harvested. Cytoplasmic extracts were prepared, and Luc activities were determined. Luc activity is expressed as degree of induction above the basal E-selectin Luc or CMV-Luc expression. Data are means ± SE; n = 4-6 for each condition.

TNF-alpha -induced ROS production mediates NF-kappa B activation. To address whether NAC regulates E-selectin promoter activity by preventing TNF-alpha -induced ROS-dependent activation of NF-kappa B, we performed electrophoretic mobility shift assays using oligonucleotides E-selectin NF-kappa BA (35 bp) or E-selectin NF-kappa BB (30 bp) containing the E-selectin promoter NF-kappa B sites 1-3. Incubation of these oligonucleotides with the nuclear extracts prepared from TNF-alpha -stimulated HPAE cells revealed the presence of two closely migrating bands corresponding to p65 homodimers (slow-migrating band) and a heterodimeric mixture of p65 and p50 (fast- migrating band; Fig. 7, A and B, lane 2, and Ref. 38). The DNA binding activity of NF-kappa B was more pronounced with E-selectin NF-kappa BB compared with E-selectin NF-kappa BA, consistent with previous findings (38). It should be noted that the NF-kappa B complex interaction may be influenced by subtle differences in sequence (18). Because NF-kappa B site 3 (GGGGATTTCC) in E-selectin NF-kappa BB probe differs from the consensus NF-kappa B sequence (GGGACTTTCC) in two bases shown in italics compared with NF-kappa B site 1 (GGATATTCCC) and site 2 (GGGAAAGTTT) in E-selectin NF-kappa BA probes, which differ in four and five bases, respectively, from the consensus NF-kappa B sequence, the difference in the binding activities and the binding patterns between NF-kappa BA and NF-kappa BB is not surprising. In competition experiments, these binding activities were effectively competed by the presence of excess unlabeled wild-type E-selectin NF-kappa BA or E-selectin NF-kappa BB oligonucleotide in a dose-dependent manner (Fig. 7, A and B, lanes 3 and 4) or by the presence of wild-type Igkappa B oligonucleotides containing NF-kappa B consensus sequences (Fig. 7, A and B, lane 7). However, the bands were not eliminated by the presence of either an irrelevant probe or an oligonucleotide bearing mutations in NF-kappa B binding sites (Fig. 7, A and B, lanes 5 and 6).


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Fig. 7.   TNF-alpha induction of NF-kappa B activity. Gel mobility shift assays were performed as described in MATERIALS AND METHODS. Nuclear extracts were prepared from HPAE cells treated for 1 h with TNF-alpha (100 U/ml) or left untreated and incubated in the absence (lane 2) or presence of indicated molar excess of cold wild-type (wt) (lanes 3 and 4) or mutant (mut)-E-selectin NF-kappa BA (lane 5), Oct-1 DNA (lane 6), or wt Igkappa B consensus DNA (lane 7) before the addition of radiolabeled wt E-selectin NF-kappa BA (A) and E-selectin NF-kappa BB (B) probes.

Pretreatment of cells for 0.5 h with NAC inhibited TNF-alpha induction of NF-kappa B activity (Fig. 8, A and B). Because NAC can also serve as a reducing agent, we evaluated the effect of a reducing agent, DTT, on TNF-alpha activation of NF-kappa B. Because NF-kappa B binding was more pronounced on NF-kappa B site 3 (Figs. 7B and 8B), we used only E-selectin NF-kappa BB probe in this experiment. At a lower concentration of 1 mM, DTT potentiated the TNF-alpha -induced NF-kappa B binding to the E-selectin promoter (Fig. 8C). At a higher concentration of 2 mM, DTT reduced the slow-migrating binding activity, which was associated with an increase in the fast-migrating binding activity to E-selectin promoter (Fig. 8C). These results indicate that NAC and DTT have differential effects on TNF-alpha induction of NF-kappa B activity, and thus the effect of NAC in preventing E-selectin expression could not be ascribed to its reducing activity.


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Fig. 8.   NAC prevents TNF-alpha -induced NF-kappa B binding to E-selectin promoter. Gel mobility shift assays were performed as described in MATERIALS AND METHODS. HPAE cells were pretreated with NAC (30 mM; A and B) or dithiothreitol (DTT; 1 and 2 mM; C) for 0.5 h and then challenged with TNF-alpha (100 U/ml). After 1 h, nuclear extracts were prepared, and their DNA binding activities were assessed using radiolabeled wt E-selectin NF-kappa BA (A), E-selectin NF-kappa BB (B), and E-selectin NF-kappa BB probes (C).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the present study, we have demonstrated that TNF-alpha -induces ROS production in HPAE cells. We present data showing that TNF-alpha -induced ROS generation serves as a critical signal in the activation of NF-kappa B and E-selectin gene expression in HPAE cells. These data include the inhibitory effects of NAC on TNF-alpha induction of 1) ROS production, 2) NF-kappa B activity, 3) E-selectin promoter activity, and 4) E-selectin mRNA and cell surface protein expression. We also demonstrated that PDTC, an antioxidant structurally unrelated to NAC, had similar effects in preventing TNF-alpha -induced ROS production and the increase in E-selectin mRNA and cell surface expression; thus the effects of NAC are likely secondary to its antioxidant action and cannot be ascribed to its ability to increase the intracellular glutathione concentration (26). It is also unlikely that NAC exerted its inhibitory effects by interfering with TNF-alpha receptor binding, since Suzuki et al. (42) have shown that inhibition of NF-kappa B activation by NAC in Jurkat cells did not occur at the level of binding of TNF-alpha to its receptor. The possibility that NAC could directly interact with NF-kappa B to interfere with its DNA binding activity can also be ruled out. If this is the case, NAC should prevent the activation of NF-kappa B irrespective of whether it occurs by an oxidant-dependent or -independent mechanism. Moynagh et al. (28) showed that IL-1beta activation of NF-kappa B in human glial cells did not involve ROS, and the response was insensitive to NAC. Moreover, NAC did not prevent the ROS generation-independent NF-kappa B activation induced by the inhibitors of serine/threonine protein phosphatases 1 and 2A, calyculin A, and okadaic acid (42). Therefore, the inhibitory effect of NAC in preventing NF-kappa B activation cannot be attributed to a direct interaction of NAC with NF-kappa B. Another possibility is that NAC selectively stimulates the rate of E-selectin mRNA degradation; however, this does not seem to be the case, since pretreatment with NAC, which prevents the TNF-alpha induction of E-selectin transcript, slightly induced E-selectin mRNA expression, which was undetectable in control untreated cells (Fig. 3, A and C). Finally, it is unlikely that reducing activity of NAC was responsible for the inhibitory action of NAC, since DTT, a reducing agent, failed to prevent the TNF-alpha -induced activation of NF-kappa B.

The present data indicate that downregulation of transcriptional activity of E-selectin promoter is the likely basis of the NAC-mediated reduction in E-selectin mRNA expression. This assertion is supported by a number of observations. In gel shift assays, NAC prevented TNF-alpha -induced binding of NF-kappa B to the E-selectin promoter. Furthermore, in transient transfection studies, we found that inhibition of E-selectin promoter-directed Luc expression correlated with suppression of E-selectin mRNA expression (maximal inhibition of ~90%; compare Figs. 3B and 6A). In control experiments, however, NAC failed to inhibit CMV-promoter-directed Luc expression, indicating that the inhibitory effect of NAC on E-selectin promoter activity was not secondary to Luc mRNA/protein degradation.

Faruqi et al. (13) have reported that the NAC-mediated inhibition of E-selectin gene transcription in IL-1beta -activated human umbilical vein endothelial cells (HUVEC) did not involve inhibition of NF-kappa B binding to E-selectin promoter, whereas NAC notably inhibited NF-kappa B binding to VCAM-1 promoter. These results are surprising since the E-selectin promoter NF-kappa B binding site (GGGATTTCCC) used in this study is identical to at least one of the two NF-kappa B binding sites in the VCAM-1 promoter (GGGTTTCCC and GGGATTTCC). Thus the basis for the different result from the present study is not clear; it may reflect differences in activation of NF-kappa B and other transcription factors that may cooperate to mediate E-selectin expression in HUVEC compared with HPAE cells.

Marui et al. (24) and others (44) have reported that the oxidant-regulated pathway controlling cytokine-induced VCAM-1 expression is distinct from E-selectin expression. Given that p65 homodimer or p50-p65 heterodimer activates transcription of these genes (11, 38, 39, 45), it is plausible that a common redox-sensitive mechanism regulates expression of both VCAM-1 and E-selectin in cytokine-activated endothelial cells. However, the sensitivity to oxidants in regulation of E-selectin and VCAM-1 expression may be different due to the following factors. First, the 5' regulatory region of the gene encoding VCAM-1 has two versus three functional NF-kappa B binding sites in the E-selectin gene (17, 38). Second, induction of these genes in response to cytokines requires participation of a specific repertoire of transcription factors in concert with NF-kappa B; that is, interferon regulatory factor-1 (31) is required for maximal VCAM-1 induction, whereas full cytokine responsiveness of E-selectin gene requires activation of high-mobility group protein-1 (Y) (21), activating transcription factor-2, and c-Jun (36). Third, antioxidants can differentially activate transcription factors; for example, PDTC induces activation of a number of transcription factors, including AP-1 (25, 29, 30). In support of this concept, we demonstrated that, as in the case of VCAM-1 (24), PDTC prevented the expression of E-selectin mRNA in a concentration-dependent manner (Fig. 4). However, unlike VCAM-1 (23), the expression of E-selectin mRNA became insensitive to PDTC after a 2-h exposure period (data not shown). Upon extended exposure, PDTC itself induced E-selectin transcript in a time-dependent fashion, although weakly and with much slower kinetics compared with the TNF-alpha response (data not shown). Munoz et al. (29) have also recently reported that PDTC mediated a delayed induction of intercellular adhesion molecule-1 mRNA expression through the activation of AP-1. These results suggest that PDTC initially affects a component that is common in the expression of these adhesion molecules, most likely activation of NF-kappa B, whereas extended exposure influences another step required for expression of E-selectin but not of VCAM-1. Because AP-1 activation is required for maximal expression of E-selectin in response to TNF-alpha (36) and because PDTC also induces AP-1 activation in endothelial cells, these findings are consistent with the concept that PDTC initially inhibits NF-kappa B activation and hence E-selectin expression, but this effect is masked at later time points because of the ability of PDTC to cooperate with TNF-alpha in inducing E-selectin expression through the activation of AP-1.

In summary, the present data indicate that TNF-alpha induces NF-kappa B activation and the resultant E-selectin gene expression by a pathway that involves formation of ROS and that can be inhibited by the antioxidant actions of NAC or PDTC. The results support the hypothesis that generation of ROS by inflammatory cytokines in endothelial cells is a critical signaling mechanism mediating E-selectin expression. Pharmacological manipulation of the redox equilibrium in endothelial cells may provide novel means of preventing activation of the E-selectin gene and hence of inappropriate endothelial hyperadhesiveness after TNF-alpha stimulation.

    ACKNOWLEDGEMENTS

We thank Dr. Vijai Baichwal of Tularik for E-selectin-luciferase plasmid and Asma Naqvi for technical assistance.

    FOOTNOTES

A. Rahman and J. Kefer contributed equally to this work.

This work was supported by National Heart, Lung, and Blood Institute Grants HL-27016, HL-46350, and HL-45638.

Present addresses: M. Bando, Dept. of Pulmonary Medicine, Jichi Medical School, Tochigi, Japan 329-04; W. D. Niles, Aurora Biosciences Corporation, 11010 Torreyana Rd., San Diego, CA 92121.

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. §1734 solely to indicate this fact.

Address for reprint requests: A. Rahman, Dept. of Pharmacology (M/C 868), The Univ. of Illinois, College of Medicine, 835 South Wolcott Ave., Chicago, IL 60612-7343.

Received 30 March 1998; accepted in final form 13 May 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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