Activation of NF-kappa B induced by H2O2 and TNF-alpha and its effects on ICAM-1 expression in endothelial cells

Andrea L. True, Arshad Rahman, and Asrar B. Malik

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Reactive oxygen species have been proposed to signal the activation of the transcription factor nuclear factor (NF)-kappa B in response to tumor necrosis factor (TNF)-alpha challenge. In the present study, we investigated the effects of H2O2 and TNF-alpha in mediating activation of NF-kappa B and transcription of the intercellular adhesion molecule (ICAM)-1 gene. Northern blot analysis showed that TNF-alpha exposure of human dermal microvascular endothelial cells (HMEC-1) induced marked increases in ICAM-1 mRNA and cell surface protein expression. In contrast, H2O2 added at subcytolytic concentrations failed to activate ICAM-1 expression. Challenge with H2O2 also failed to induce NF-kappa B-driven reporter gene expression in the transduced HMEC-1 cells, whereas TNF-alpha increased the NF-kappa B-driven gene expression ~10-fold. Gel supershift assay revealed the presence of p65 (Rel A), p50, and c-Rel in both H2O2- and TNF-alpha -induced NF-kappa B complexes bound to the ICAM-1 promoter, with the binding of the p65 subunit being the most prominent. In vivo phosphorylation studies, however, showed that TNF-alpha exposure induced marked phosphorylation of NF-kappa B p65 in HMEC-1 cells, whereas H2O2 had no effect. These results suggest that reactive oxygen species generation in endothelial cells mediates the binding of NF-kappa B to nuclear DNA, whereas TNF-alpha generates additional signals that induce phosphorylation of the bound NF-kappa B p65 and confer transcriptional competency to NF-kappa B.

redox state; intercellular adhesion molecule-1 promoter; tumor necrosis factor-alpha ; oxidants; nuclear factor-kappa B; signaling; hydrogen peroxide


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

REACTIVE OXYGEN SPECIES (ROS) play a critical role in the mechanism of inflammation associated with septicemia and acute respiratory distress syndrome (17). ROS can function as second messengers in the signal transduction pathways activated by the proinflammatory cytokines interleukin-1beta and tumor necrosis factor (TNF)-alpha (28, 31). These cytokines regulate the redox-sensitive transcription factor nuclear factor (NF)-kappa B, which participates in a variety of immune, inflammatory, and acute-phase responses (4, 6). NF-kappa B/Rel transcription factors are composed of five distinct DNA binding subunits: NF-kappa B1 (p50), NF-kappa B2 (p100/p52), Rel A (p65), Rel B, and c-Rel (23). Of these, p65, Rel B, and c-Rel possess both transactivation and DNA binding domains, whereas p50 and p52 lack the transactivation domain and serve primarily as DNA binding subunits (36). The different family members can associate in homodimer or heterodimer combinations through the highly conserved NH2-terminal NF-kappa B/Rel/Dorsal or Rel homology domain (16). NF-kappa B dimers are most commonly composed of p65 and p50 or p52 subunits (3, 39). Inactive NF-kappa B dimers are sequestered in the cytosol in association with inhibitory molecules of the Ikappa B family. Stimulation of cells with TNF-alpha results in phosphorylation of Ikappa B-alpha on serine-32 and -36 or of Ikappa B-beta on serine-19 and -23 by Ikappa B kinases alpha  and beta  (13, 46). This targets Ikappa B-alpha and Ikappa B-beta for polyubiquitination and proteasome-mediated degradation (11, 43). Release from Ikappa B unmasks the nuclear localization signal of NF-kappa B and thus mediates its translocation to the nucleus (7).

Although phosphorylation of Ikappa B is a critical regulatory step in the activation of NF-kappa B, studies (8, 36) suggested that phosphorylation of NF-kappa B p65 after its release from Ikappa B may enhance its transactivation potential. Thus phosphorylation of NF-kappa B may be an additional mechanism regulating the expression of NF-kappa B-dependent genes. In the present study, we addressed the activation of such a gene, intercellular adhesion molecule-1 (ICAM-1), an inducible cell surface glycoprotein belonging to the immunoglobulin supergene family (42). Interaction of ICAM-1 with its counterreceptors (leukocyte beta 2-integrins CD11a/CD18 and CD11b/CD18) is a requirement for the recruitment and extravasation of leukocytes to sites of tissue injury and infection (40, 41). ICAM-1 mRNA and cell surface expression are upregulated in response to a variety of inflammatory mediators including TNF-alpha (18, 22, 33).

Rahman and colleagues (31, 32) have shown that TNF-alpha induces ROS generation in endothelial cells through a protein kinase (PK) C-dependent mechanism and that this event is critical in signaling the activation of NF-kappa B and the genes encoding ICAM-1 and E-selectin. Studies (35, 37) have also shown that H2O2 mediates TNF-alpha -induced NF-kappa B activation; however, there are reports (10, 21) that ROS may not signal the activation of NF-kappa B and NF-kappa B-dependent gene expression. A study (20) has shown that H2O2 induced the expression of ICAM-1 mRNA in a human dermal microvessel endothelial cell (HMEC-1) line; in other studies, H2O2 failed to induce DNA binding activities of the transcription factors activator protein (AP)-1 and NF-kappa B (21) that are known to activate ICAM-1 gene transcription in endothelial cells (18, 22, 26). In light of these observations, we compared the effects of H2O2 with those of TNF-alpha in mediating the activation of NF-kappa B in endothelial cells using HMEC-1 cells. We demonstrated that both H2O2 and TNF-alpha induced the DNA binding activity of NF-kappa B; however, the binding of NF-kappa B induced by H2O2 was transcriptionally "silent" as determined by its inability to activate the NF-kappa B-driven reporter gene and ICAM-1 expression. In contrast, the NF-kappa B binding activity induced by TNF-alpha was transcriptionally active. These data suggest that ROS production signals nuclear NF-kappa B DNA binding activity; however, the bound NF-kappa B may remain transcriptionally inactive. Thus TNF-alpha activates additional signaling pathway(s) that induce phosphorylation of NF-kappa B p65 and confer transcriptional competency to the DNA-bound NF-kappa B.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Human dermal microvascular endothelial cells (HMEC-1) were maintained in culture in MCDB-131 medium (GIBCO BRL, Life Technologies, Gaithersburg, MD) supplemented with 5% fetal bovine serum (FBS), 10 ng/ml of human epidermal growth factor (Sigma, St. Louis, MO), 1 µg/ml of hydrocortisone, 5 mg/ml of L-glutamine, and antibiotics (penicillin-streptomycin) ("complete" MCDB-131). HMEC-1 cells, an immortalized endothelial cell line transformed by the SV40 large T antigen, retain the endothelial cell phenotype and functional characteristics; they express and secrete von Willebrand factor, take up acetylated low-density lipoprotein, form tubes when grown in Matrigel, and express CD31, CD35, ICAM-1 (CD54), and CD44 (1). HMEC-1 cells were passaged in uncoated culture dishes until they reached confluence. The cells between passages 17 and 21 were used in these studies, and all cell studies were carried out under the same conditions. Confluent HMEC-1 cells were serum starved for 2-4 h, washed two times with serum- and phenol red-free Dulbecco's modified Eagle's medium, allowed to equilibrate for 30 min, and then incubated with the indicated concentrations of H2O2 (Sigma) or recombinant human TNF-alpha (specific activity 2.3 × 107 U/mg protein; Promega, Madison, WI) for all experiments unless otherwise specified.

Cell viability. Two methods were used to evaluate cell viability after H2O2 and TNF-alpha challenge. Trypan blue (Sigma) exclusion studies were carried out according to manufacturer's suggested protocol. Confluent cells were treated with H2O2 or TNF-alpha for 2 h, washed gently with PBS two times, trypsinized, resuspended, and washed with complete MCDB-131. The cell suspension (10 µl) was mixed with 10 µl of 1× trypan blue solution, and 10 µl of the resulting mixture were loaded onto a hemocytometer. Results showed that 95-98% of the cells were viable after challenge with 100 µM H2O2 or 100 U/ml of TNF-alpha , whereas only 70-80% of the cells were viable after 1 mM H2O2 challenge. Reduction of tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was also used to assay cell viability (19). These results showed that HMEC-1 cells exposed to 100 µM H2O2 were viable after treatment for 5, 60, or 120 min, whereas cells challenged with 1 mM H2O2 were not (Lum H, personal communication).

Intracellular oxidant generation. Oxidant generation in HMEC-1 cells was measured as previously described (32). Briefly, confluent HMEC-1 cells were challenged with TNF-alpha or H2O2 for 15 min in serum-free phenol red-free DMEM, and all subsequent steps were conducted in the dark. After challenge, the cells were washed two times with MCDB-131 (2% FBS) and loaded for 20 min with 1 µM 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate bis(acetoxymethyl ester) (C-DCHF-DA; Molecular Probes, Eugene, OR). The dye solution was freshly prepared in prewarmed MCDB-131 (2% FBS) for each sample, and the samples were independently stained so that they were exposed to dye for the same amount of time. After dye loading at 37°C, the cells were rinsed two times with MCDB-131 (2% FBS) containing no dye, and the cultures were viewed with a fluorescence microscope and photographed. Quantitative fluorescence was imaged with a Nikon Diaphot 200 microscope (Nikon, Garden City, NJ) and ImagePro Plus software (Media Cybernetics, Silver Spring, MD).

Northern analysis. Total RNA was isolated from HMEC-1 cells with QIAGEN RNeasy columns after homogenization through QIAshredder columns (QIAGEN, Chatsworth, CA). Quantification and purity of RNA were assessed by the ratio of absorbance at 260 nm to that at 280 nm. An aliquot of RNA (10-20 µg) was analyzed for ICAM-1 mRNA expression as previously described (30). Autoradiography was performed with an intensifying screen at -70°C for 12-24 h.

Flow cytometric analysis. HMEC-1 monolayers on six-well tissue culture dishes were left untreated or were stimulated with H2O2 or TNF-alpha as indicated, washed two times with cold PBS (Ca2+ and Mg2+ free), removed by trypsinization, and incubated in 20% horse serum for 30 min at 4°C to block nonspecific binding. Samples were pelleted at 4°C and washed two times with cold 3% horse serum in PBS. The cells were incubated in the same wash buffer with the anti-human ICAM-1 monoclonal antibody RR1/1 (5-10 µg/ml) (provided by Dr. Robert Rothlein, Boehringer Ingelheim, Ridgefield, CT) (14), washed two times, and incubated with a FITC-conjugated goat anti-mouse secondary antibody (10-100 µg/ml; Sigma). After two washes and fixation with 2% paraformaldehyde while being vortexed, the samples were analyzed by flow cytometry in a FACScan cytofluorometer (Becton Dickinson, Mountain View, CA), with mean fluorescence intensity gated above that of the secondary antibody alone. When purified mouse IgG (1 mg/ml) was used in place of RR1/1, no significant increase in fluorescence was observed.

Reporter gene construct, endothelial cell transfection, and luciferase assay. The NF-kappa B-luciferase plasmid (pNF-kappa B-Luc) containing five copies of the consensus NF-kappa B sequence linked to the minimal E1B promoter-luciferase reporter gene was purchased from Stratagene (La Jolla, CA). HMEC-1 cells were plated on six-well tissue culture dishes and transfected the following day at ~70-80% confluence according to the DEAE-dextran method with slight modifications (12). Briefly, 2 µg of DNA were mixed with 0.15 mg/ml of DEAE-dextran and 0.1 mM chloroquine in serum- and antibiotic-free MCDB-131 for 1.5 h followed by a 30-s shock with 10% dimethyl sulfoxide in PBS. The cells were then washed two times with complete MCDB-131 containing 10% FBS and grown to confluence. To determine transfection efficiency, HMEC-1 cells were transfected with the plasmid pGreen Lantern-1, which expresses green fluorescence protein (GIBCO BRL), and the transfected cells were then subjected to flow cytometry to determine green fluorescence protein expression. Using the above protocol, we showed that transfection efficiencies were ~50%, with <5% variability in a given sample. After transfection, the cells were exposed to H2O2 or TNF-alpha at the indicated concentrations and time periods. Cell extracts were prepared, and luciferase activity was determined with a TD 20/20 luminometer (Turner Designs, Sunnyvale, CA) with the Promega Biotech luciferase reporter assay system. Values are expressed as relative light units per microgram of protein extract, and the protein content was determined with the Bio-Rad (Hercules, CA) protein determination kit.

Nuclear protein isolation. Cells were washed three times with ice-cold Tris-buffered saline (TBS) and resuspended in 400 µl of buffer A [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 µg/ml of leupeptin, and 5 µg/ml of aprotonin]. After 15 min, Nonidet P-40 (NP-40) was added to a final concentration of 0.6%. Nuclei were pelleted and suspended in 50 µl of buffer C [20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 5 µg/ml of leupeptin, and 5 µg/ml of aprotonin]. After 30 min of agitation at 4°C, the lysates were centrifuged, and the supernatants containing the nuclear proteins were transferred to fresh vials in 15-µl aliquots and stored at -70°C until used.

Electrophoretic mobility shift assay. Electrophoretic mobility shift assay (EMSA) was performed as previously described (31). Briefly, nuclear extract (10 µg) was incubated with 1 µg of poly(dI-dC) in 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. End-labeled, double-stranded oligonucleotides (30,000 counts/min each) were then added in the absence and presence of a 25- to 100-fold molar excess of cold wild-type (ICAM-1NF-kappa B) or mutant (mut-ICAM-1NF-kappa B) competitor and incubated for 15 min at room temperature. In antibody supershift experiments, nuclear extracts were incubated for 15 min at room temperature with polyclonal rabbit antibody to various NF-kappa B proteins (2 µg/20 µl; Santa Cruz Biotechnology, Santa Cruz, CA) before incubation with the labeled probe for another 15 min at room temperature. The DNA-protein complexes were resolved by 5-6% native polyacrylamide gel electrophoresis (PAGE) in low ionic strength buffer (0.25× Tris-borate-EDTA). Oligonucleotides used for the gel shift analysis were 5'-AGCTTGGAAATTCCGGAGCTG-3' for ICAM-1NF-kappa B and 5'-AGCTTccAAATTCCGGAGCTG-3' for mut-ICAM-1NF-kappa B. The oligonucleotide designated as ICAM-1NF-kappa B represents the 21-bp sequence of ICAM-1 promoter encompassing the downstream NF-kappa B binding site located 223 bp upstream from the transcription initiation site (18, 22). The oligonucleotide mut-ICAM-1NF-kappa B is similar to ICAM-1NF-kappa B except that it has 2-bp mutations in the NF-kappa B binding site. NF-kappa B sequence motifs within the oligonucleotides are underlined and mutations are shown in lowercase. In separate experiments, nuclear proteins were incubated with the oligonucleotide Ig-kappa B (5'-AGTTGAGGGGACTTTCCCAGGC-3'), which contains the consensus NF-kappa B binding site present in the Ig gene and pNF-kappa B-Luc construct.

In vivo labeling of cells and immunoprecipitation. In vivo labeling of cells and immunoprecipitation were carried out as described by Ollivier et al. (27) with slight modifications. Briefly, confluent monolayers of HMEC-1 cells on six-well tissue culture dishes were washed two times with phosphate-free medium and incubated for 2 h before being loaded with 200-500 µCi [32P]orthophosphate/ml for 3-4 h, followed by stimulation with TNF-alpha or H2O2. Cells were needle lysed in 500 µl of cold radioimmunoprecipitation assay buffer [1% Triton X, 1% deoxycholate, 150 mM NaCl, 10 mM Tris · HCl (pH 7.5), 1 mM EGTA, 1 mM PMSF, 1 mM Na3VO4, 0.1% SDS, 5 µg/ml of aprotonin, and 1 nM calyculin A]. NF-kappa B p65 was recovered by immunoprecipitation (after preclearance with 50 µl of protein A agarose beads) with an anti-p65 antibody (1 µg, 4-18 h at 4°C; Santa Cruz Biotechnology) and protein A agarose beads (2 h at 4°C; Boehringer Mannheim, Indianapolis, IN). The precipitated proteins were washed three times, including one high salt wash (radioimmunoprecipitation assay with 500 mM NaCl), and were then pelleted. The antibody-p65-bead complexes were boiled in sample buffer containing 25 mM DTT for 5 min and spun, and the supernatants were separated on 10-12.5% SDS-polyacrylamide gels for 4.5 h (20 mA). Coomassie blue staining of the gels revealed the presence of a single band corresponding to a molecular mass of 65 kDa. The gels were then either dried and visualized by autoradiography or transferred to polyvinylidene difluoride membranes for Western blotting and confirmation of NF-kappa B p65.

Western blot analysis. The immunoprecipitated NF-kappa B p65 samples were subjected to SDS-PAGE (10%) as described in In vivo labeling of cells and immunoprecipitation and then transferred to polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked for 1 h with 5% (wt/vol) nonfat dry milk solution in 10 mM Tris base, 150 mM NaCl, and 0.05% Tween 20 before the membranes were incubated for 1 h with rabbit polyclonal anti-human NF-kappa B p65 antibodies (Santa Cruz Biotechnology) diluted 1:1,000. The membranes were washed three times with TBS-Tween 20 and incubated for another hour with goat anti-rabbit horseradish peroxidase-linked IgG (Amersham, Arlington Heights, IL) diluted 1:5,000. After the washes, antibody-labeled proteins were detected by the enhanced chemiluminescence method (Amersham) according to manufacturer's recommendations.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

H2O2 failed to mimic TNF-alpha -induced expression of ICAM-1 in HMEC-1 cells. We compared the effects of H2O2 with those of TNF-alpha on NF-kappa B-dependent ICAM-1 gene expression. Northern blot analysis showed that H2O2 failed to activate ICAM-1 transcription in HMEC-1 cells at all concentrations studied, whereas TNF-alpha induced robust ICAM-1 mRNA expression (Fig. 1). HMEC-1 cells were subjected to fluorescence-activated cell-sorting analysis with the anti-ICAM-1 monoclonal antibody RR1/1 (14) to determine whether H2O2 altered the cell surface expression of ICAM-1 independent of increased mRNA expression. As shown in Fig. 2, H2O2 failed to induce ICAM-1 cell surface expression. In contrast, TNF-alpha induced a marked increase in ICAM-1 cell surface expression within 2 h, which increased further after 18 h of exposure (Fig. 2).


View larger version (55K):
[in this window]
[in a new window]
 
Fig. 1.   Effects of tumor necrosis factor (TNF)-alpha and H2O2 on the induction of intercellular adhesion molecule (ICAM)-1 mRNA expression. Confluent monolayers of human dermal microvascular endothelial cells (HMEC-1) were stimulated for 1 h with (+) and without (-) H2O2 or TNF-alpha (A) or at the indicated concentrations of H2O2 and TNF-alpha (B). Total RNA was isolated, and equivalent amounts were subjected to Northern blot analysis with a human ICAM-1 cDNA probe. Ethidium bromide staining of 28S rRNA is shown to indicate equal loading. Results are representative of 3 separate experiments.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 2.   H2O2 failed to mimic TNF-alpha -induced cell surface ICAM-1 expression. Confluent HMEC-1 cells were left untreated (control) or stimulated with H2O2 (100 µM) or TNF-alpha (100 U/ml) for the indicated time periods. ICAM-1 cell surface expression was quantitated by flow cytometry with the primary monoclonal antibody against human ICAM-1 (RR1/1) and a FITC-conjugated secondary antibody. Results are representative of 2 separate experiments performed in duplicate.

TNF-alpha and H2O2 challenges induced similar intracellular oxidant generation. To determine whether differences in ICAM-1 induction by H2O2 and TNF-alpha could be ascribed to differences in the oxidant-generating capacity of the mediators, confluent HMEC-1 cells were challenged with TNF-alpha (100 U/ml) or H2O2 (subcytolytic concentrations ranging from 10 to 500 µM) for 15 min and loaded with the oxidant-sensitive dye C-DCHF-DA as described in METHODS. After incorporation and subsequent cleavage by cellular esterases, C-DCHF-DA was trapped in cells and converted to the fluorescent species carboxydichlorofluorescein after oxidation. Results showed that 100 U/ml of TNF-alpha and 100 and 500 µM H2O2 both induced comparable C-DCHF-DA fluorescence compared with that in control cells, whereas the fluorescence activated by 10 µM H2O2 was similar to the control values (Fig. 3A). Figure 3B shows the quantification of the relative fluorescence intensity of each field shown in Fig. 3A. Total relative fluorescence for each image was divided into three classes of brightness where class 1 represents the area of each cell with lowest brightness intensity and class 3 represents the area of each cell with highest brightness intensity. Control cells exhibited fluorescence in brightness class 1, and treatment with 100 and 500 µM H2O2 or 100 U/ml of TNF-alpha caused a shift to a higher brightness, with maximum fluorescence occurring in brightness class 2 (Fig. 3B). These results indicate that differences in ICAM-1 expression are not secondary to a differential oxidant-generating capacity of the two mediators.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 3.   TNF-alpha and H2O2 induced similar intracellular oxidant generation. Confluent HMEC-1 cells were loaded with 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate bis(acetoxymethyl ester) (C-DCHF-DA) as described in METHODS and then were left untreated or stimulated with H2O2 or TNF-alpha at the indicated concentrations for 15 min. Oxidant generation was measured by C-DCHF-DA fluorescence. A: fluorescent images of representative control and H2O2- and TNF-alpha -treated cells (representative of 2 separate experiments performed in triplicate). B: relative fluorescence intensities for each condition in A were determined, compiled, and partitioned into 3 brightness classes, with class 1 representing the lowest fluorescence and class 3 representing the highest fluorescence. The relative fluorescence intensity for cells stimulated with 100 and 500 µM H2O2 and with 100 U/ml of TNF-alpha was shifted to the higher intensity classes compared with control and 10 µM H2O2-treated cells.

H2O2 failed to mimic TNF-alpha -induced transcriptional activity of NF-kappa B. We next evaluated the ability of H2O2 to induce transcriptional activation of NF-kappa B because oxidants generated in response to TNF-alpha are critical in signaling NF-kappa B activation and ICAM-1 (31). We transiently transfected HMEC-1 cells with the plasmid pNF-kappa B-Luc containing five copies of the consensus NF-kappa B site linked to a minimal adenovirus E1B promoter-luciferase reporter gene. NF-kappa B-directed luciferase activity increased ~10-fold when the transfected cells were exposed to TNF-alpha (Fig. 4). In contrast, H2O2 failed to induce NF-kappa B-directed luciferase expression (Fig. 4). Because H2O2 failed to induce transcriptional activation of pNF-kappa B-Luc, we focused our attention on NF-kappa B to explain the basis for the lack of ICAM-1 induction.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   H2O2 failed to mimic TNF-alpha -induced transcriptional activity of nuclear factor (NF)-kappa B. HMEC-1 cells were transfected with the pNF-kappa B-Luc construct containing 5 copies of NF-kappa B linked to a firefly luciferase reporter gene. After 24-48 h, cells were exposed to H2O2 and TNF-alpha at the indicated concentrations. Cell extracts were prepared 6 h after treatment and assessed for luciferase activity [expressed as relative light units (RLU)/µg cell protein]. Data are means ± SE; n = 3-6 extracts/condition.

H2O2 and TNF-alpha induced NF-kappa B DNA binding activity. Because the antioxidant actions of N-acetylcysteine and pyrrolidine dithiocarbamate prevented TNF-alpha -induced NF-kappa B DNA binding activity (31, 32), we determined whether the inability of H2O2 to activate ICAM-1 transcription could be explained by the failure of NF-kappa B to bind to the ICAM-1 promoter. We performed the EMSA using the oligonucleotide ICAM-1NF-kappa B (21 bp), which contains the downstream NF-kappa B sequence in the ICAM-1 promoter required for ICAM-1 expression (18, 22). Both H2O2 and TNF-alpha induced marked binding of NF-kappa B to the ICAM-1 promoter (Fig. 5A). These results also showed that H2O2 induced NF-kappa B binding in a time-dependent manner, with the maximum response occurring after 30-60 min (Fig. 5B).


View larger version (70K):
[in this window]
[in a new window]
 
Fig. 5.   H2O2 and TNF-alpha induced NF-kappa B binding to ICAM-1 promoter. Nuclear proteins were isolated from HMEC-1 cells after treatment and subjected to electrophoretic mobility shift assay (EMSA) as described in METHODS. A: confluent monolayers were left untreated (control) or were challenged with 100 µM H2O2 or 100 U/ml of TNF-alpha for 45 min. Samples were resolved by 5.5% PAGE. B: HMEC-1 cells were treated with H2O2 to determine the time course of DNA binding activity. Samples were resolved by 5.0% PAGE. C: nuclear proteins from the 30-min H2O2-treated cells shown in B were incubated in the absence (lanes 1 and 6) and presence of a 25- or 100-fold molar excess of unlabeled wild-type (wt; lanes 2 and 3, respectively) or mutant (mut; lanes 4 and 5, respectively) ICAM-1NF-kappa B oligonucleotide before the addition of radiolabeled wt ICAM-1NF-kappa B (lanes 1-5) or mut ICAM-1NF-kappa B (lane 6) probe. Samples were resolved by 5.0% PAGE. Results are representative of 3 separate experiments.

We next determined the specificity of NF-kappa B binding activity. Competition experiments were performed in which a 25- and a 100-fold molar excess of unlabeled ICAM-1NF-kappa B oligonucleotide were incubated with the labeled ICAM-1NF-kappa B oligonucleotide. Excess unlabeled ICAM-1NF-kappa B quenched the binding of NF-kappa B to the labeled ICAM-1 promoter sequence in a concentration-dependent manner, with greater inhibition in the presence of a 100-fold molar excess of oligonucleotide (Fig. 5C, lanes 2 and 3). In contrast, incubation of labeled ICAM-1NF-kappa B with excess unlabeled mutant oligonucleotide (mut-ICAM-1NF-kappa B; 2-bp mutation in ICAM-1NF-kappa B) had little effect on NF-kappa B binding (Fig. 5C, lanes 4 and 5). The small reduction in NF-kappa B binding in the presence of excess mut-ICAM-1NF-kappa B oligonucleotide (Fig. 5C, lane 5) can be ascribed to the binding of NF-kappa B to the mutant oligonucleotide (Fig. 5C, lane 6). Taken together, these data indicate that H2O2 induces the specific binding of NF-kappa B to the ICAM-1 promoter in a manner similar to the binding induced by TNF-alpha (31).

Because the consensus NF-kappa B sequence of pNF-kappa B-Luc differs from the NF-kappa B sequence present in the ICAM-1 promoter, we also performed an EMSA using an oligonucleotide that contains this sequence to determine whether H2O2 can induce the binding of NF-kappa B to this site. As shown in Fig. 6, both H2O2 (lanes 2 and 3) and TNF-alpha (lane 4) caused the binding of NF-kappa B to the consensus sequence. Thus the failure of H2O2 to induce NF-kappa B-dependent reporter gene activation and ICAM-1 expression as shown above is independent of the NF-kappa B DNA binding activity induced by H2O2.


View larger version (62K):
[in this window]
[in a new window]
 
Fig. 6.   H2O2 and TNF-alpha induced binding to the consensus NF-kappa B sequence. Confluent HMEC-1 monolayers were left untreated or were treated for 60 min with H2O2 or TNF-alpha , and nuclear proteins were isolated. Equivalent amount (10 µg) of each sample was incubated with the radiolabeled consensus Ig-kappa B oligonucleotides for 15 min and subjected to EMSA as described in METHODS. Samples were resolved by 5.5% PAGE. Results are representative of 2 separate experiments.

H2O2- and TNF-alpha -induced NF-kappa B complexes were predominantly composed of NF-kappa B p65. The gel supershift assay was carried out to determine whether differences in the H2O2 and TNF-alpha responses could be explained by differences in the NF-kappa B complex bound to the ICAM-1 promoter. Antibody directed against p50 (which lacks the transactivation domains), p65, or c-Rel (which contain the transactivation domains) were incubated with nuclear extracts isolated from H2O2- or TNF-alpha -treated cells before addition of the labeled ICAM-1NF-kappa B oligonucleotide. Antibody to p65 significantly reduced both H2O2 (Fig. 7A, lane 4)- and TNF-alpha (Fig. 7, A, lane 6, and B, lanes 6 and 7)-induced NF-kappa B binding to the ICAM-1 promoter. In contrast, antibodies against p50 and c-Rel had less effect on DNA binding activities (Fig. 7, A, lanes 2, 3, and 7, and B, lanes 4 and 5). Therefore, differential binding of NF-kappa B subunits to DNA induced by H2O2 and TNF-alpha fails to explain the observed differences in the transcriptional activation responses induced by the two mediators.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 7.   H2O2- and TNF-alpha -activated NF-kappa B complexes were predominantly composed of p65 subunit. A: nuclear proteins were isolated from HMEC-1 cells stimulated for 45 min with either H2O2 or TNF-alpha . Equivalent amount (10 µg) of each sample was incubated with 2 µg of rabbit antibodies specific for p50 (alpha -p50; lane 2), p65 (alpha -p65; lanes 4 and 6), or c-Rel (alpha -c-Rel; lanes 3 and 7) for 15 min at room temperature before addition of radiolabeled wt ICAM-1NF-kappa B oligonucleotide. Samples were resolved by 5.5% PAGE. B: nuclear proteins isolated from HMEC-1 cells treated with TNF-alpha for 60 min (lanes 2-7) were incubated with alpha -p50 (lanes 4 and 5) or alpha -p65 (lanes 6 and 7) as in A. Samples were resolved by 6.0% PAGE. The relative optical density represents the intensity of NF-kappa B complexes bound to the ICAM-1 promoter. Results are representative of 3 separate experiments.

TNF-alpha and H2O2 differentially phosphorylated NF-kappa B p65 subunit. Because phosphorylation of NF-kappa B p65 may regulate transcriptional activation of NF-kappa B (2, 45), we determined whether alterations in the phosphorylation status of NF-kappa B p65 after H2O2 and TNF-alpha stimulation could explain the differential response. Cells were loaded with [alpha -32P]orthophosphate in phosphate-free medium and stimulated with H2O2 or TNF-alpha for 30 min. Immunoprecipitation of NF-kappa B p65 followed by SDS-PAGE showed marked phosphorylation of p65 in response to TNF-alpha (Fig. 8A, lane 3) but not to H2O2 (Fig. 8A, lane 2). Similar results were obtained after 15 min of H2O2 or TNF-alpha challenge (data not shown). To rule out the possibility that the phosphorylated proteins observed did not represent coimmunoprecipitation of other NF-kappa B subunits associated with p65, Western blot analysis was performed with the anti-p65 antibody. These results indicated the selective recovery of p65 after immunoprecipitation (Fig. 8B).


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 8.   TNF-alpha and H2O2 differentially phosphorylated the NF-kappa B p65 subunit (p65-P). A: 32P-labeled HMEC-1 cells were left unchallenged (lane 1) or were challenged with H2O2 (lane 2) or TNF-alpha (lane 3) for 30 min. Cells were lysed, and p65 was recovered by immunoprecipitation. Samples were resolved by 12.5% SDS-PAGE. Gels were dried, and autoradiography was performed. Nos. at left, molecular mass. B: aliquots of p65 from H2O2- or TNF-alpha -treated cells recovered in A were resolved by 10% PAGE, transferred to polyvinylidene difluoride membranes, and subjected to Western blot analysis with the antibody directed against p65 as described in METHODS. The relative intensity of the p65-P band in A demonstrates the selective phosphorylation of NF-kappa B p65 induced by TNF-alpha . Results are representative of 3 separate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Oxidants have been proposed to function as second messengers in the activation of transcription factors (5, 24, 28). Studies have shown that H2O2 induces the activation of NF-kappa B and the transcription of NF-kappa B-dependent genes in several cell types (37). Although oxidants can induce binding of NF-kappa B in endothelial cells (5), it remains unclear whether additional signals are involved in the transcriptional activation of NF-kappa B-dependent genes such as ICAM-1.

Oxidant generation induced by TNF-alpha in endothelial cells contributes to the mechanism of NF-kappa B activation and transcription of the genes encoding ICAM-1 and E-selectin (31, 32). In the present study, we determined whether H2O2 mimics the effects of TNF-alpha in mediating the activation of NF-kappa B and ICAM-1 expression. We demonstrated that H2O2 promoted the binding of the NF-kappa B p65 subunit to its cognate site; interestingly, the bound NF-kappa B remained transcriptionally "silent." In contrast, the TNF-alpha -induced binding of NF-kappa B p65 promoted a robust NF-kappa B-driven reporter gene activation and ICAM-1 expression. The results suggest that the transcriptional activity of NF-kappa B induced by TNF-alpha is conferred by phosphorylation of the NF-kappa B p65 subunit, whereas H2O2 has no effect on the phosphorylation of this subunit. Thus phosphorylation of NF-kappa B p65 appears to be critical in mediating the transcriptional activation of NF-kappa B-dependent genes in endothelial cells.

H2O2 is a permeable oxidant that rapidly enters cells but is also rapidly degraded by intracellular antioxidants. To ensure that intracellular antioxidants did not scavenge H2O2 and interfere with the response, we made studies in which HMEC-1 cells transfected with pNF-kappa B-Luc were exposed to freshly prepared H2O2 every 2 h for up to a 6-h period. Replenishment of H2O2 in these studies failed to induce transcriptional activity of NF-kappa B (data not shown); thus the present results cannot be explained on the basis of degradation of H2O2.

There may be various reasons for the failure of H2O2 to activate NF-kappa B-dependent transcription in HMEC-1 cells. One possibility is that H2O2 inhibits NF-kappa B-dependent transcription by oxidizing the critical cysteine residue required for NF-kappa B DNA binding activity (25). We observed that H2O2 was capable of promoting NF-kappa B DNA binding activity but that the bound NF-kappa B was transcriptionally inactive; thus under the conditions of our experiment, oxidation of NF-kappa B does not appear to be responsible for failure of H2O2 to induce NF-kappa B activity. Another possibility is that there may be differences in the redox state resulting from differences in oxidant generation. To address this question, endothelial cells were loaded with the oxidant-sensitive dye C-DCHF-DA with which we assayed oxidant generation in response to H2O2 and TNF-alpha . Because challenge of cells with H2O2 and TNF-alpha resulted in comparable dye fluorescence, any difference in the redox state cannot explain the failure of H2O2 to activate NF-kappa B in HMEC-1 cells. Another possibility is that H2O2 and TNF-alpha differentially activate the mitogen-activated protein kinase (MAPK) pathway regulating NF-kappa B-dependent gene expression (44). However, in other studies, we have shown that both H2O2 and TNF-alpha induced comparable and rapid phosphorylation of MAPK in HMEC-1 cells (True A, Lum H, Beno DW, and Malik AB, unpublished results); thus differences in activation of this pathway also cannot explain the present observations.

The question arises as to what confers transcriptional competency to the DNA-bound NF-kappa B after TNF-alpha challenge. Previous studies (22, 38) showed that overexpression of NF-kappa B p65 in endothelial cells transactivated the ICAM-1 and vascular cell adhesion molecule-1 promoters in a kappa B site-dependent manner. Phosphorylation of serine-529 in the transactivation domain 1 of NF-kappa B p65 in HeLa cells exposed to TNF-alpha may promote NF-kappa B transcriptional activation (45). Phosphorylation at this residue was specific in that it did not interfere with nuclear translocation or DNA binding activity. Phosphorylation of transactivation domain 2 of NF-kappa B in phorbol ester-stimulated COS-7 cells also resulted in enhanced NF-kappa B transcriptional activity (36). In the present study, we observed that the TNF-alpha -challenged cells contained the phosphorylated p65 species, consistent with a critical role for NF-kappa B p65 phosphorylation in mediating NF-kappa B activation. The kinase responsible for p65 phosphorylation in endothelial cells is unknown. Casein kinase-II and MAPKs have been proposed to phosphorylate p65 in vitro (8, 44). PKC-zeta may also regulate phosphorylation and transcriptional activity of NF-kappa B p65 in endothelial cells (2) and thus may mediate ICAM-1 gene transcription after TNF-alpha stimulation (31). In addition, phosphorylation of NF-kappa B p65 by the catalytic subunit of PKA may activate NF-kappa B (47). NF-kappa B p65 phosphorylation may also be necessary for association with the coactivator p300/cAMP-responsive element binding protein (CBP), a requirement for NF-kappa B transactivation (15, 29). PKA-induced phosphorylation of NF-kappa B p65 exposed the binding site for interaction with p300/CBP (48), suggesting a mechanism by which p65 phosphorylation can induce transcriptional activation. Thus failure of phosphorylation of NF-kappa B p65 by one or more of these kinases could explain the inability of H2O2 to induce NF-kappa B gene expression in HMEC-1 cells.

Another explanation for our results is that H2O2 fails to activate transcription factors that may coordinately regulate transcription of the ICAM-1 gene. For example, CAAT enhancer binding protein (C/EBP) was reported to cooperate with NF-kappa B in the mechanism of TNF-alpha -induced ICAM-1 expression (18). We showed that H2O2 did not induce transcriptionally active NF-kappa B in cells transfected with the pNF-kappa B-Luc construct. Because this construct is driven by NF-kappa B sequences and C/EBP does not bind to these sequences, it is unlikely that the failure of H2O2 to activate C/EBP can explain our observations. The role of AP-1 was also proposed in the mechanism of ICAM-1 transcription (26), although the ability of H2O2 to activate AP-1 in endothelial cells remains controversial (21). The results obtained with pNF-kappa B-Luc reporter construct argue that transcriptionally silent NF-kappa B is the more likely explanation for the absence of ICAM-1 induction by H2O2. A recent study (9) has shown that p50 can act as a negative regulator of NF-kappa B-dependent gene expression. Because we did not find that H2O2 preferentially induced p50 binding, it is unlikely that H2O2 prevented the transcriptional activation of NF-kappa B and expression of ICAM-1 by this mechanism.

The present observations differ from those in previous reports (20, 34). The cell culture conditions may have been different such that there may be differences in the redox state of cells. This could result in oxidant-induced activation of NF-kappa B and, thereby, ICAM-1 expression. The present results are important because they unmask a phosphorylation-dependent mechanism that may signal transcriptional activation of NF-kappa B after NF-kappa B binding to DNA.

Although H2O2 did not induce transcriptionally active NF-kappa B, the present results are fully consistent with our observations that TNF-alpha -induced ICAM-1 expression can be blocked by antioxidants (31). Because the results point to an important role for oxidants generated by TNF-alpha in signaling the binding of NF-kappa B to the promoter, it would be expected that antioxidants would prevent TNF-alpha -induced NF-kappa B binding and the resultant ICAM-1 expression as demonstrated (31).

In summary, the present results show that H2O2 induces the nuclear DNA binding activity of NF-kappa B and that the binding of NF-kappa B induced by H2O2 alone is insufficient to activate NF-kappa B-dependent reporter gene activation and ICAM-1 transcription. These results suggest that the H2O2-induced DNA binding activity of NF-kappa B in the absence of phosphorylation of NF-kappa B p65 fails to activate ICAM-1 gene transcription. Thus TNF-alpha -induced phosphorylation of NF-kappa B p65 may be an important factor regulating expression of the NF-kappa B-dependent ICAM-1 gene in endothelial cells.


    ACKNOWLEDGEMENTS

We thank Tina Leisner for assistance with fluorescence-activated cell-sorting analysis and Khandaker N. Anwar for technical support.


    FOOTNOTES

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

Address for reprint requests and other correspondence: A. Rahman, Dept. of Pharmacology, College of Medicine, Univ. of Illinois, 835 South Wolcott Ave., Chicago, IL 60612-7343 (E-mail: ARahman{at}uic.edu).

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.

Received 23 February 2000; accepted in final form 28 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ades, EW, Candal FJ, Swerlick RA, George VG, Summers S, Bosse DC, and Lawley TJ. Establishment of an immortalized endothelial cell line. J Invest Dermatol 99: 683-690, 1992[Abstract].

2.   Anrather, J, Csizmadia V, Soares MP, and Winkler H. Regulation of NF-kappa B RelA phosphorylation and transcriptional activity by p21(ras) and protein kinase Czeta in primary endothelial cells. J Biol Chem 274: 13594-13603, 1999[Abstract/Free Full Text].

3.   Baeuerle, PA, and Baltimore D. NF-kappa B: ten years after. Cell 87: 13-20, 1996[ISI][Medline].

4.   Baeuerle, PA, and Henkel T. Function and activation of NF-kappa B in the immune system. Annu Rev Immunol 12: 141-179, 1994[ISI][Medline].

5.   Barchowsky, AS, Munro R, Morana SJ, Vincenti MP, and Treadwell M. Oxidant-sensitive and phosphorylation-dependent activation of NF-kappa B and AP-1 in endothelial cells. Am J Physiol Lung Cell Mol Physiol 269: L829-L836, 1995[Abstract/Free Full Text].

6.   Barnes, PJ, and Karin M. NF-kappa B: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 336: 1066-1071, 1997[Free Full Text].

7.   Beg, AA, Ruben SM, Scheinman RI, Haskill S, Rosen CA, and Baldwin JAS I kappa B interacts with the nuclear localization sequences of the subunits of NF-kappa B: a mechanism for cytoplasmic retention. Genes Dev 10: 1899-1913, 1992.

8.   Bird, TA, Schooley K, Dower SK, Hagen H, and Virca GD. Activation of nuclear transcription factor NF-kappa B by interleukin-1 is accompanied by casein kinase II-mediated phosphorylation of the p65 subunit. J Biol Chem 272: 32606-32612, 1997[Abstract/Free Full Text].

9.   Bouhuslav, J, Kravchenko VV, Parry GCN, Erlich JH, Gerondakis S, Mackman N, and Ulevitch RJ. Regulation of an essential innate immune response by the p50 subunit of NF-kappa B. J Clin Invest 102: 1645-1652, 1998[Abstract/Free Full Text].

10.   Bradley, JR, Johnson DR, and Pober JS. Endothelial activation by hydrogen peroxide: selective increases of intercellular adhesion molecule-1 and major histocompatibility complex class 1. Am J Pathol 142: 1598-1609, 1993[Abstract].

11.   Chen, ZJ, Parent L, and Maniatis T. Site-specific phosphorylation of Ikappa Balpha by a novel ubiquitination-dependent protein kinase activity. Cell 84: 853-862, 1996[ISI][Medline].

12.   Cullen, BR. Use of eukaryotic expression technology in functional analysis of cloned genes. Methods Enzymol 152: 684-704, 1987[ISI][Medline].

13.   DiDonato, JA, Hayakawa DM, Rothwarf DM, Zandi E, and Karin MA. A Cytokine-responsive Ikappa B kinase that activates the transcriptional factor NF-kappa B. Nature 338: 548-554, 1997.

14.   Dustin, ML, Rothlein R, Bhan AK, Dinarello CA, and Springer TA. Induction by IL-1 and interferon-gamma : tissue distribution, biochemistry and function of a natural adherence molecule (ICAM-1). J Immunol 137: 245-254, 1986[Abstract/Free Full Text].

15.   Gerritsen, ME, Williams AJ, Neish AS, Moore S, Shi Y, and Collins T. CREB-binding protein/p300 are transcriptional coactivators of p65. Proc Natl Acad Sci USA 94: 2927-2932, 1997[Abstract/Free Full Text].

16.   Grimm, S, and Baeuerle PA. The inducible transcription factor NF-kappa B: structure-function relationship of its protein subunits. Biochem J 290: 297-308, 1993[ISI][Medline].

17.   Halliwell, B, and Gutteridge JMC Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol 186: 1-85, 1989.

18.   Hou, J, Baichwal V, and Cao Z. Regulatory elements and transcription factors controlling basal and cytokine-induced expression of the gene encoding ICAM-1. Proc Natl Acad Sci USA 91: 11641-11645, 1994[Abstract/Free Full Text].

19.   Hussain, RF, and Nouri AME A new approach for measurement of cytotoxicity using colorimetric assay. J Immunol Methods 100: 89-96, 1993.

20.   Lakshminarayanan, V, Beno DW, Costa RH, and Roebuck KA. Differential regulation of interleukin-8 and intercellular adhesion molecule-1 by H2O2 and tumor necrosis factor-alpha in endothelial and epithelial cells. J Biol Chem 272: 32910-32918, 1997[Abstract/Free Full Text].

21.   Lakshminarayanan, V, Drab-Weiss EA, and Roebuck KA. H2O2 and tumor necrosis factor-alpha induce differential binding of the redox-responsive transcription factors AP-1 and NF-kappa B to the interleukin-8 promoter in endothelial and epithelial cells. J Biol Chem 273: 32670-32678, 1998[Abstract/Free Full Text].

22.   Ledebur, HC, and Parks TP. Transcriptional regulation of the intercellular adhesion molecule-1 gene by inflammatory cytokines in human endothelial cells: essential roles of a variant NF-kappa B site and p65 homodimers. J Biol Chem 270: 933-943, 1995[Abstract/Free Full Text].

23.   Liou, CC, and Baltimore D. Regulation of the NF-kappa B/rel transcription factor and I kappa B inhibitor system. Curr Opin Cell Biol 5: 466-487, 1993.

24.   Lo, Y, and Cruz TF. Involvement of reactive oxygen species in cytokine and growth factor induction of c-fos in chondriocytes. J Biol Chem 270: 11727-11730, 1995[Abstract/Free Full Text].

25.   Matthews, JR, Wakasugi N, Virelizier JL, Yodi J, and Hay RT. Thioredoxin regulates the DNA binding activity of NF-kappa B by reduction of a disulphide bond involving cysteine 62. Nucleic Acids Res 20: 3821-3830, 1992[Abstract].

26.   Munoz, C, Castellanos MC, Alfranca A, Vara A, Estaban MA, Redonodo JM, and de Landazuri MO. Transcriptional up-regulation of intercellular adhesion molecule-1 in human endothelial cells by the antioxidant pyrrolidine dithiocarbamate involves the activation of activating protein-1. J Immunol 15: 3587-3597, 1996.

27.   Ollivier, V, Parry GCN, Cobb RR, de Prost D, and Mackman N. Elevated cyclic AMP inhibits NF-kappa B-mediated transcription in human monocytic cells and endothelial cells. J Biol Chem 271: 20828-20835, 1996[Abstract/Free Full Text].

28.   Pahl, HL, and Baeuerle PA. Oxygen and the control of gene expression. Bioessays 16: 479-501, 1994.

29.   Perkins, ND, Felzien LK, Betts JC, Leung K, Beach DH, and Nabel GJ. Regulation of NF-kappa B by cyclin-dependent kinases associated with the p300 coactivator. Science 275: 523-527, 1997[Abstract/Free Full Text].

30.   Rahman, A, Anwar KN, True AL, and Malik AB. Thrombin-induced p65 homodimer binding to downstream NF-kappaB site of the promoter mediates endothelial ICAM-1 expression and neutrophil adhesion. J Immunol 162: 5466-5476, 1999[Abstract/Free Full Text].

31.   Rahman, A, Bando M, Kefer J, Anwar KN, and Malik AB. Protein kinase C-activated oxidant generation in endothelial cells signals intercellular adhesion molecule-1 gene transcription. Mol Pharmacol 55: 575-583, 1999[Abstract/Free Full Text].

32.   Rahman, A, Kefer J, Bando M, Niles WD, and Malik AB. E-selectin expression in human endothelial cells by TNF-alpha -induced oxidant generation and NF-kappa B activation. Am J Physiol Lung Cell Mol Physiol 275: L533-L544, 1998[Abstract/Free Full Text].

33.   Rahman, A, Roebuck KA, and Malik AB. Transcriptional regulation of endothelial adhesion molecule gene expression by oxidants and cytokines. In: Nitric Oxide and Radicals in the Pulmonary Vasculature, edited by Weir EK, Archer SL, and Reeves HT.. New York: Futura, 1996, p. 63-85.

34.   Roebuck, KA, Rahman A, Lakshminarayanan V, Janakidevi K, and Malik AB. H2O2 and tumor necrosis factor-alpha activate intercellular adhesion molecule 1 (ICAM-1) gene transcription through distinct cis-regulatory elements within the ICAM-1 promoter. J Biol Chem 270: 18966-18974, 1995[Abstract/Free Full Text].

35.   Schmidt, KN, Amstad P, Cerutti P, and Baeuerle PA. The roles of hydrogen peroxide and superoxide as messengers in the activation of transcription factor NF-kappa B. Chem Biol 2: 13-22, 1995[ISI][Medline].

36.   Schmitz, ML, dos Santos Silva MA, and Baeuerle PA. Transactivation domain 2 (TA2) of p65 NF-kappa B; similarity to TA1 and phorbol ester-stimulated activity and phosphorylation in intact cells. J Biol Chem 270: 15576-15584, 1995[Abstract/Free Full Text].

37.   Schreck, R, Albermann K, and Baeuerle PA. Nuclear factor kappa B: an oxidative stress-responsive transcription factor of eukaryotic cells. Free Radic Res Commun 17: 221-237, 1992[ISI][Medline].

38.   Shu, HB, Agranoff AB, Nabel EG, Leung K, Duckett CS, Neish AS, Collins T, and Nabel GJ. Differential regulation of vascular cell adhesion molecule 1 gene expression by specific NF-kappa B subunits in endothelial and epithelial cells. Mol Cell Biol 13: 6283-6289, 1993[Abstract].

39.   Siebenlist, U, Franzoso G, and Brown K. Structure, regulation and function of NF-kappa B. Annu Rev Cell Biol 10: 405-455, 1994[ISI].

40.   Smith, CW, Marlin SD, Rothlein R, Toman C, and Anderson DC. Cooperative interactions of LFA-1 and Mac-1 with intercellular adhesion molecule-1 facilitating adherence and transendothelial migration of human neutrophils in vitro. J Clin Invest 83: 2008-2017, 1989[ISI][Medline].

41.   Smith, CW, Rothlein R, Hughes BJ, Mariscalo MM, Rudloff HE, Schmalsteig FC, and Anderson DC. Recognition of an endothelial determinant for CD 18-dependent human neutrophil adherence and transendothelial migration. J Clin Invest 82: 1746-1756, 1988[ISI][Medline].

42.   Staunton, DE, Marlin SD, Stratowa C, Dustin ML, and Springer TA. Primary structure of ICAM-1 demonstrates interaction between members of immunoglobulin and integrin supergene families. Cell 54: 925-933, 1988.

43.   Traenckner, EB, Pahl HL, Henkel T, Schmidt KN, Wilk S, and Baeuerle PA. Phosphorylation of human Ikappa B on serine residues 32 and 36 controls Ikappa B proteolysis and NF-kappa B activation in response to diverse stimuli. EMBO J 14: 2876-2883, 1995[Abstract].

44.   Vanden Berghe, W, Plaisance S, Boone E, De Bosscher K, Schmitz ML, Fiers W, and Haegeman G. p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways are required for NF-kappa B p65 transactivation mediated by tumor necrosis factor. J Biol Chem 273: 3285-3290, 1998[Abstract/Free Full Text].

45.   Wang, D, and Baldwin AS, Jr. Activation of NF-kappa B-dependent transcription by tumor necrosis factor-alpha is mediated through phosphorylation of RelA/p65 on serine 529. J Biol Chem 273: 29411-29416, 1998[Abstract/Free Full Text].

46.   Zandi, E, Rothwarf DM, Delhase M, Hayakawa M, and Karin M. The Ikappa B kinase complex (IKK) contains two kinase subunits, IKKalpha and IKKbeta , necessary for Ikappa B phosphorylation and NF-kappa B activation. Cell 91: 243-252, 1997[ISI][Medline].

47.   Zhong, H, SuYang H, Erdjument-Bromage H, Tempst P, and Ghosh S. The transcriptional activity of NF-kappaB is regulated by the Ikappa B-associated PKAc subunit through a cyclic AMP-independent mechanism. Cell 89: 413-424, 1997[ISI][Medline].

48.   Zhong, H, Voll RE, and Ghosh S. Phosphorylation of NF-kappa B p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. Mol Cell 1: 661-671, 1998[ISI][Medline].


Am J Physiol Lung Cell Mol Physiol 279(2):L302-L311
1040-0605/00 $5.00 Copyright © 2000 the American Physiological Society