©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
HO and Tumor Necrosis Factor- Activate Intercellular Adhesion Molecule 1 (ICAM-1) Gene Transcription through Distinct cis-Regulatory Elements within the ICAM-1 Promoter (*)

(Received for publication, May 9, 1995; and in revised form, June 7, 1995 )

Kenneth A. Roebuck (1) (2),  (§),  (¶),   Arshad Rahman (1)(§),   Venkatesh Lakshminarayanan (1) Kilambi Janakidevi (3) Asrar B. Malik (1)

From the (1)Departments of Pharmacology and (2)Immunology/Microbiology, Rush-Presbyterian-St. Luke's Medical Center/Rush Medical College, Chicago, Illinois 60612 and the (3)Department of Physiology and Cell Biology, Albany Medical College, Albany, New York 12208

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We investigated the mechanisms by which H(2)O(2) increases intercellular adhesion molecule 1 (ICAM-1; CD54) expression in endothelial cells. The H(2)O(2)-induced increase in ICAM-1 mRNA was inhibited by actinomycin D, by the antioxidant N-acetylcysteine, and by 3-aminobenzamide (which blocks oxidant-induced AP-1 activity), but not by pyrrolidine dithiocarbamate (which blocks oxidant-induced NF-kappaB activity). Nuclear run-on and transient transfections of ICAM-1 promoter constructs indicated that H(2)O(2) stimulated ICAM-1 gene transcription by activation of a distinct region of the ICAM-1 promoter. The H(2)O(2)-responsive element was localized to sequences between -981 and -769 (relative to the start codon). Located within this region are two 16-base pair repeats, each containing binding sites for the transcription factors AP-1 and Ets. A similar composite AP-1/Ets element isolated from the macrophage scavenger receptor gene conferred H(2)O(2) responsiveness to a minimal promoter. Mutation of the 16-base pair repeats within the ICAM-1 promoter prevented H(2)O(2)-induced DNA binding activity, and their deletion abrogated the H(2)O(2)-induced transcriptional activity. In contrast, TNFalpha induced ICAM-1 transcription via activation of promoter sequences between -393 and -176, a region with C/EBP and NF-kappaB binding sites. The results indicate that H(2)O(2) activates ICAM-1 transcription through AP-1/Ets elements within the ICAM-1 promoter, which are distinct from NF-kappaB-mediated ICAM-1 expression induced by TNFalpha.


INTRODUCTION

Adhesion of circulating polymorphonuclear leukocytes (PMN) (^1)to the vascular endothelium is a critical step in the inflammatory response (Nourshargh and Williams, 1990). PMN adhesion to the endothelium occurs during reperfusion of tissues when reactive oxygen intermediates such as H(2)O(2) are generated (Hernandez et al., 1987). The adhesion event is mediated by molecules present or expressed on the surface of endothelial cells and PMN (Lo et al., 1989). Endothelial cells express intercellular adhesion molecule 1 (ICAM-1; CD54), a counter-receptor for CD11/CD18 integrin (Dustin et al., 1988) that promotes adhesion and transendothelial migration of PMN (Smith et al., 1989). Studies using monoclonal antibodies show that increased cell surface ICAM-1 expression is required for migration of PMN to sites of inflammation and PMN-mediated endothelial injury associated with reperfusion (Kukielka et al., 1993). ICAM-1 gene expression is induced by tumor necrosis factor-alpha (TNFalpha), interferon , and interleukin-1beta (Myers et al., 1992; Wertheimer et al., 1992; Look et al., 1994).

Recent studies show that the reactive oxidant, H(2)O(2), also promotes ICAM-1 expression in endothelial cells and ICAM-1-dependent adhesion of PMN (Lo et al., 1993; Bradely et al., 1993; Sellak et al., 1994). H(2)O(2) was recently reported to also increase ICAM-1 expression on keratinocytes (Ikeda et al., 1994). In human umbilical vein endothelial cells (HUVEC), we found that oxidant-induced ICAM-1 expression was associated with increased ICAM-1 mRNA levels occurring 1 h after H(2)O(2) exposure (Lo et al., 1993). H(2)O(2) activates transcription factors, AP-1 and NF-kappaB, in a mouse osteoblastic cell line (Nose et al., 1991) and in HeLa and Jurkat cells (Meyer et al., 1993). The ICAM-1 gene contains a number of AP-1-like and NF-kappaB-like binding sites within its promoter region (Voraberger et al., 1991). Taken together, these observations suggest that the activation of these transcription factors by H(2)O(2) may be a mechanism of endothelial ICAM-1 gene expression.

In this study, we examined the basis of H(2)O(2)-induced ICAM-1 expression in endothelial cells. We showed that H(2)O(2) activated ICAM-1 gene transcription via a 212-base pair (bp) promoter region between 981 and 769 bp upstream of the coding sequences. This region contained two 16-bp repeats which are binding sites for the transcription factors AP-1 and Ets. AP-1/Ets composite elements were shown to be sufficient to mediate H(2)O(2)-induced transcription. Although the AP-1/Ets elements also responded to TNFalpha, the TNFalpha-induced ICAM-1 expression was mediated by promoter sequences between 393 and 176 bp upstream of the gene, containing binding sites for C/EBP and NF-kappaB. Therefore, H(2)O(2) and TNFalpha activate ICAM-1 gene transcription in endothelial cells through distinct cis-regulatory elements within the ICAM-1 promoter. The results identify a novel oxidant response element and indicate that mediator-specific regulation of ICAM-1 expression involves the interaction of multiple factors with the ICAM-1 promoter.


EXPERIMENTAL PROCEDURES

Materials

Diethylpyrocarbonate, DMEM, heparin, HEPES, 3.0% H(2)O(2), MOPS, PMSF, spermidine, spermine, pyrrolidine dithiocarbamate (PDTC), and N-acetylcysteine (N-Cys(Ac)) were purchased from Sigma. We purchased 3-aminobenzamide (3-AB) from Pfaltz and Bauer (Stamford, CT). Guanidine thiocyanate, restriction enzymes, random primer labeling kit, QuikHyb hybridization mix, and Duralose-UV nitrocellulose membranes were purchased from Stratagene. Human ICAM-1 cDNA was provided by Dr. T. Springer, Harvard Medical School, Boston, MA. Plasmid containing the cDNA for rRNA was provided by Dr. M. L. Brown, Boehringer-Ingelheim Pharmaceuticals, Ridgefield, CT. Plasmid EL1-BS, containing partial human E-selectin cDNA, was provided by Dr. L. Osborn, Biogen, Cambridge, MA. Agarose, actinomycin D, LipofectAMINE, and RPMI were purchased from Life Technologies, Inc. Riboprobe Gemini System II and RNase-free DNase were purchased from Promega Biotech. Fetal bovine serum was obtained from Hyclone Laboratories. [alpha-P]dCTP (3,000 Ci/mmol), [-P]ATP (3,000 Ci/mmol), and [alpha-P]UTP (3,000 Ci/mmol) were purchased from DuPont NEN. The antisense oligomer ISIS 1570 (5`-TGGGAGCCATAGCGAGGCTGA-3`) to the 5` end of the ICAM-1 cDNA and a nonsense oligomer (5`-AGTCGGAGCGATACCGAGGGT-3`) were generous gifts from Sterling-Winthrop Drug Co., Rensselaer, NY. Oligonucleotides were purchased from Integrated DNA Technologies Inc. (Coralville, IA). ICAM-1 luciferase reporter gene plasmids were gifts from Dr. C. Stratowa, Vienna, Austria.

Cell Cultures

Human umbilical vein endothelial cells (HUVEC) at the first passage were purchased from Clonetics Corp. (San Diego, CA). HUVEC were grown on fibronectin-coated flasks or plates in RPMI medium containing 10-20% fetal calf serum, 6.5 µg/ml endothelial-derived growth factor from bovine neural tissue, and 75 µg/ml heparin. All experiments used cells under the eighth passage. EAhy926 cells, a hybrid cell line of HUVEC and A549 cell line (derived from human lung epithelial type II cells), was provided by Dr. Edgell (University of North Carolina, Chapel Hill) and cultured as described (Edgell et al., 1983). EAhy926 cells retain endothelial cell morphology and express the endothelial cell-specific marker human factor VIII-related antigen (Edgell et al., 1983). EAhy926 cells were maintained in DMEM-high glucose, in 5% CO(2), 10% fetal calf serum, and passaged by removal in trypsin-EDTA buffer (0.14 M NaCl, 2.68 mM KCl, 0.42 mM NaH(2)PO(4), 0.012 M NaHCO(3), 0.01 M dextrose, 0.05% trypsin, 0.53 mM EDTA).

Confluent cells were washed twice with serum-free DMEM (without phenol red) containing 20 mM HEPES and incubated for 2 h before treatment with the agents described below. The experiments using the inhibitors (PDTC, N-Cys(Ac), or 3-AB) required a 1-h preincubation period in serum-free medium with each inhibitor, and treatment was continued during the 1-h H(2)O(2) exposure period.

Reporter Gene Constructs, Transfections, and Luciferase Assays

The ICAM-1 LUC reporter plasmid and its 5` deletion derivatives have been described previously (Voraberger et al., 1991). The full-length ICAM-1 promoter construct contains approximately 1.4 kb of ICAM-1 5`-flanking DNA linked to the firefly luciferase (LUC) gene. Transfection into cells showed that this ICAM-1 construct was responsive to phorbol 12-myristate 13-acetate and TNFalpha (Voraberger et al., 1991). The macrophage scavenger receptor constructs containing three copies of the AP-1/Ets element or mutations of the element linked to luciferase have been described previously (Wu et al., 1994). Cells were plated 24 h prior to transfection at 5 10^5 cells per 6-cm plate. The cells were refed with fresh medium containing 10% fetal calf serum 4 h before lipofection (Malone et al., 1989) with LipofectAMINE as described by Life Technologies, Inc. Transfection of 100-mm plates at 80% confluency typically contained 8 µg of reporter plasmid (ICAM-1 LUC) and 2 µg of beta-gal expression plasmid DNA. The cells were transfected for 5 to 14 h. After a recovery period, the cells were divided into five 35-mm plates. At 24 h before treatment with H(2)O(2) (concentration range 100-400 µM), TNFalpha (100 units/ml) or phorbol 12-myristate 13-acetate (50 ng/ml), the cells were incubated in medium containing 0.5% fetal calf serum. The cell extract was prepared and assayed for luciferase activity using Promega Biotec assay systems and beta-galactosidase activity using the Tropix (Bedford, MA) assay system. Protein content was determined using a Bio-Rad protein determination kit. Mean luciferase activity per µg of protein extract was normalized to the beta-galactosidase activity (which in control experiments was not affected by H(2)O(2)).

RNA Isolation and Northern Analysis

All solutions used for RNA analysis were treated with diethylpyrocarbonate (0.1%) and sterilized or prepared in sterile diethylpyrocarbonate-treated water. Glassware was baked at 240 °C for a minimum of 4 h to remove traces of RNase. Total RNA was isolated according to the procedure of Chomczynski and Sacchi(1987). Medium was removed, and the endothelial cell layer was rinsed with ice-cold, Ca -and Mg-free phosphate-buffered saline (PBS), and lysed in acid guanidine thiocyanate. The lysate was drawn through a 26-gauge needle and extracted with acid phenol/chloroform (5:1). After a 30-60-min incubation on ice, the mixture was centrifuged for 30 min at 12,000 g. The aqueous phase was collected and RNA was precipitated with equal volume of ice-cold isopropyl alcohol. After allowing the RNA to precipitate for 1 h at -70 °C, RNA was pelleted by centrifugation for 30 min at 12,000 g. The RNA pellet was washed twice with 75% ethanol, briefly dried, and dissolved in 0.5% SDS in diethylpyrocarbonate-treated water. Quantification and purity of RNA were assessed by A/A absorption, and RNA samples with ratios above 1.9 were used for further analysis.

The RNA samples (20 µg/lane) were subjected to gel electrophoresis in denaturing 1% formaldehyde-agarose gels and transferred overnight in 20 SSC (3 M sodium chloride, 0.3 M sodium citrate, pH 7.0) to Duralose-UV nitrocellulose membranes. The membranes were baked for 2 h in vacuo at 80 °C to fix the RNA. Blots were prehybridized for 30 min at 68 °C in QuikHyb solution and hybridized for 2 h at 68 °C with random-primed P-labeled probes. After hybridization, the blots were washed twice for 15 min each at room temperature in 2 SSC with 0.1% SDS followed by 2 washes for 30 min each at 60 °C in 0.1 SSC with 0.1% SDS. The washed blots were exposed to Hybond film (Amersham) for 12 to 48 h at -70 °C using an intensifying screen. The signal intensities were quantified by scanning the autoradiograms with the Beckman R112 densitometer. All blots were hybridized with P-labeled probes of ICAM-1 cDNA (0.96-kb SalI to PstI fragment) and glyceraldehyde-3-phosphate dehydrogenase (1.1 kb PstI fragment). Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control for RNA loading and normalized by densitometry of the ICAM-1 signal.

For treatment of HUVEC with antisense and nonsense oligonucleotides, the cells were rinsed as described above with serum-free DMEM and incubated for 4 h in serum-free DMEM medium with addition of 5 µg/ml Lipofectin and an oligonucleotide at concentrations of 50 and 100 nM. After incubation, the medium was removed, fresh medium containing Lipofectin and the particular oligonucleotide was added, and the cells were treated for 1 h with 100 µM H(2)O(2). RNA was isolated and processed for Northern analysis.

Nuclear Run-on Assay

Nuclei were isolated from HUVEC (3-5 10^7) according to the procedure described by Clayton and Darnell(1983). Cells were washed twice with cold PBS, harvested by scraping, and centrifuged for 10 min at 1,000 rpm. The cell pellet was washed once with ice-cold PBS and once with RSB buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, and 3.0 mM MgCl(2)). The cells were suspended in 10 ml of RSB and incubated on ice for 10 min. The cell pellets were collected by centrifugation, when the cells were sufficiently swollen as monitored by microscopy. Cell pellets were resuspended in 5 ml of RSB and homogenized with a dounce type B homogenizer. The homogenate was treated briefly with 0.1% Triton X-100 to remove cytoplasmic tags. The nuclear pellet was collected by centrifugation for 10 min at 1500 rpm at 4 °C. The nuclear pellet was resuspended in 210 µl of freezing buffer (50 mM Tris-HCl, pH 8.0, 5 mM MgCl(2), 0.5 mM dithiothreitol, and 40% glycerol), flash frozen in liquid nitrogen, and stored at -70 °C.

The nuclei were incubated for 30 min at 30 °C in 0.3 ml of the assay mixture (25 mM Tris-HCl, pH 8.0, 1.25 mM concentration each of ATP, CTP, and GTP, 12.5 mM MgCl(2), 325 mM KCl, and 250 µCi of [alpha-P]UTP). RNase-free DNase (20 µl of 2 µg/ml) was added and incubated for an additional 15 min at 30 °C. The run-on reaction was terminated by the addition of 36 µl of 10 SET buffer (10% SDS, 100 mM Tris-HCl, pH 7.5, and 50 mM EDTA). Proteinase K (100 µg) was added and incubated for 45 min at 37 °C, and the reaction mixture was extracted once with buffer-saturated phenol/chloroform (1:1) and once with chloroform/isoamyl alcohol (24:1). The aqueous phase was collected, ammonium sulfate (final concentration of 2.3 M) was added, and the RNA was precipitated with an equal volume of isopropyl alcohol. After 1 h at -70 °C, the RNA was pelleted and washed twice with 75% ethanol. The pellet was dissolved in 100 µl of TE buffer (10 mM Tris-HCl, pH 7.4, and 1 mM EDTA) and passed through a Sephadex G-50 column to remove any unincorporated nucleotides. Filters were prepared for hybridization by application of denatured plasmids (5 µg/slot) using a slot blot apparatus. Plasmids containing cDNAs for ICAM-1, E-selectin, ribosomal RNA (rRNA), and glyceraldehyde-3-phosphate dehydrogenase were used for the experiments. Baked filters were hybridized with the RNA in the run-on assay as described for Northern analysis, and autoradiograms were developed.

Nuclear Protein Extracts

HUVEC were prepared for nuclear extracts as described by Shapiro et al.(1988). Briefly, the cells were treated with H(2)O(2), TNFalpha (100 units/ml) or medium for 1 h prior to harvesting. The cells were washed twice with ice-cold PBS and collected by centrifugation (Sorvall RT6000) for 5 min at 2,000 rpm. The cell pellet was resuspended in 5 volumes of hypotonic buffer (10 mM HEPES, pH 7.9, 0.75 mM spermidine, 0.15 mM spermine, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM DTT, 10 mM KCl, 0.5 mM PMSF) and incubated on ice for 10 min to allow the cells to swell. Cells were collected by centrifugation (Sorvall RT6000) for 7.5 min at 3,000 rpm in the cold. The cell pellet was resuspended in twice the original volume of ice-cold hypotonic buffer. Cells were homogenized with 30 strokes of a Wheaton dounce glass homogenizer (pestle B), followed by the addition of one-tenth volume of restore buffer (2.2 M sucrose, 10 mM HEPES pH 7.9, 0.75 mM spermidine, 0.15 mM spermine, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM DTT, 10 mM KCl, and 0.5 mM PMSF). Nuclei were collected by centrifugation (Sorvall RC2-B) at 10,000 rpm in a Sorvall HB4 rotor in the cold for 3.5 min. The nuclei pellet was resuspended in 3 ml of nuclei lysis buffer (20 mM HEPES, pH 7.9, 0.42 M NaCl, 0.75 mM spermidine, 0.15 spermine, 0.2 mM EDTA, 0.2 mM EGTA, 2.0 mM DTT, 25% glycerol, and 0.5 mM PMSF). Nuclear debris was removed by ultracentrifugation (Beckman L8-M ultracentrifuge) at 40,000 rpm in a Beckman Ti80 fixed angle rotor for 90 min at 1 °C, and 0.33 g of finely powdered ammonium sulfate was added to each milliliter of the collected supernatant and mixed gently by rocking in the cold for 60 to 90 min until the ammonium sulfate was completely dissolved. The precipitated nuclear protein was collected by ultracentrifugation (Beckman L8-M) at 30,000 rpm in a Beckman Ti80 fixed angle rotor for 20 min at 1 °C. Nuclear protein pellets were resuspended in 200 µl of nuclear dialysis buffer (20 mM HEPES, pH 7.9, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 2.0 mM DTT, and 0.1 mM PMSF) and dialyzed twice for 90 min against 200 ml of NDB in the cold. Nuclear extracts were cleared of insoluble material by microcentrifugation for 10 min, and 30 µl of nuclear protein extracts were aliquoted and stored in a liquid nitrogen freezer until use. Protein concentrations were determined by the Bio-Rad assay kit.

Gel Mobility Shift Assay

The electrophoretic mobility shift assay was performed as described by Roebuck et al.(1993). Nuclear extracts prepared from HUVEC by the method of Shapiro et al.(1988) were incubated with 50,000 cpm (0.1 to 0.5 ng) of various P-end-labeled double-stranded synthetic deoxyoligonucleotide probes for 30 min at 25 °C in a 20-µl reaction volume containing 12% glycerol, 12 mM HEPES-NaOH (pH 7.9), 60 mM KCl, 5 mM MgCl(2), 4 mM Tris-HCl (pH 7.9), 0.6 mM EDTA (pH 7.9), 0.6 mM DTT, and 1 µg of poly(dI)bullet(dC). DNA probes were end-labeled with [-P]ATP (3,000 µCi/mmol) and T4 polynucleotide kinase. The labeled DNA probe was purified on push columns (Stratagene). Protein-DNA complexes were resolved in 5% native polyacrylamide gels pre-electrophoresed for 30-60 min at room temperature in 0.25 TBE buffer (22.5 mM Tris borate and 0.5 mM EDTA, pH 8.3). Dried gels were exposed overnight to x-ray film with an intensifying screen at -70 °C. Oligonucleotides used for the gel shift analysis were as follows: ICAM-1 AP-1/Ets, 5`-GCTGCTGCCTCAGTTTCCC-3`; ICAM-1 NF-kappaB, 5`-GCCCGGGGAGGATTCCTGGGCCCC-3`; ICAM-1 TRE, 5`-GACCGTGATTCAAGCTTA-3`; ICAM-1 AP-1 motif, 5`-TGGCCAGTGACTCGCAGCCCCAGC-3`; AP-1 m/Ets, 5`-GCTGCgtaagacGTTTCCCAGC-3`; AP-1/Ets-m, 5`-GCTGCTGCCTCAGTcagtCAGC-3`.

Sequence motifs within the oligonucleotide are underlined, the mutations are in lowercase, and the relative positions of the sequence motifs are shown in Fig.7and Fig. 8. The NF-kappaB oligonucleotide corresponds to the element upstream of the AP-3 site and downstream of the AP-1/Ets repeats.


Figure 7: H(2)O(2) activates the ICAM-1 gene promoter. A, structure of the ICAM-1 promoter luciferase reporter gene construct. Rectangles indicate the location (relative to the start site of translation) of binding sites for the transcription factors AP-1, AP-3, NF-kappaB, C/EBP, and Ets. A 12-O-tetradecanoylphorbol-13-acetate responsive element (TRE) is located at -321. The arrow downstream of the TATA box indicates the start site of translation (ATG). B, ICAM-1 promoter activity in HUVEC. The ICAM-1 LUC construct was transfected into HUVEC as described under ``Experimental Procedures.'' At 24 h post-transfection, the cells were exposed to 100, 200, or 400 µM H(2)O(2) or to 100 units/ml TNFalpha. Cells were harvested 24 h after H(2)O(2) or TNFalpha treatment, and cell extracts were assessed for luciferase activity. C, ICAM-1 promoter activity in EAhy926 cells. Cells were transfected as described under ``Experimental Procedures.'' Phorbol 12-myristate 13-acetate (50 ng/ml)- and TNFalpha (100 units/ml)-treated cells were included for comparison. Luciferase activity is expressed as relative light units (RLU)/10 s/µg of protein normalized to beta-galactosidase activity expressed by a cotransfected beta-galactosidase expression vector.




Figure 8: Localization of the H(2)O(2) responsive region of the ICAM-1 gene promoter. The structure of the different ICAM-1 promoter luciferase constructs is shown to the left. Rectangles indicate the location of various DNA binding motifs. The AP-1/Ets repeats are indicated by solid rectangles. The nucleotide position of the 5` end of each construct is given relative to the translation initiation codon of the gene. EAhy926 cells were transfected with the ICAM-1 luciferase constructs and treated with H(2)O(2) (400 µM) or TNFalpha (100 units/ml) as described under ``Experimental Procedures.'' Luciferase activity normalized to beta-galactosidase activity is expressed as mean fold increase relative to the untreated medium control of each ICAM-1 promoter construct. Results are shown as mean ± S.D. of 3 to 5 separate experiments.




RESULTS

H(2)OInduces de Novo mRNA Synthesis

We have previously reported that exposure of human umbilical vein endothelial cells (HUVEC) to 100 µM H(2)O(2) for 1 h resulted in maximal accumulation of steady-state ICAM-1 message, which could be detected as early as 30 min after oxidant exposure (Lo et al., 1993). To determine whether the H(2)O(2)-induced increase in ICAM-1 mRNA was the result of increased de novo synthesis of the message or decreased rate of message degradation, we examined the effects of actinomycin D, a RNA synthesis inhibitor. We carried out two experiments: (i) actinomycin D was added to the cells at the same time as H(2)O(2) exposure (Fig.1) and (ii) cells were first pretreated with H(2)O(2) for 1 h to maximize the expression of ICAM-1 message followed by treatment with actinomycin D to block new mRNA synthesis (Fig.2).


Figure 1: Effects of actinomycin D on H(2)O(2)-induced expression of ICAM-1 mRNA. Confluent HUVEC were treated with 50 µM (lanes 2 and 4) or 100 µM H(2)O(2) (lanes 3 and 5) for 1 h either in the absence (lanes 2 and 3) or presence (lanes 4 and 5) of actinomycin D. Total RNA was isolated and analyzed by Northern blot. Control cells (lane 1) did not receive any treatment. A, autoradiogram; B, bar graph representing the relative intensities of the ICAM-1 mRNA signals (representative of 4 separate experiments).




Figure 2: Stability of H(2)O(2)-induced ICAM-1 mRNA. HUVEC were treated with 100 µM H(2)O(2) for 1 h to achieve peak RNA synthesis (lane 2) followed by addition of actinomycin D (50 µM) for 0.5 to 2 h (lanes 3-6). Total RNA was isolated from cells at the times indicated and analyzed by Northern blot. A, autoradiogram; B, bar graph presenting the relative intensities of the ICAM-1 mRNA signals (representative of 4 separate experiments).



Treatment with actinomycin D at the time of H(2)O(2) exposure (50 µM or 100 µM) abrogated ICAM-1 message induction (Fig.1; compare lanes 2 and 3 with 4 and 5). To examine the effect of H(2)O(2) on mRNA stability, endothelial cells were first exposed to 100 µM H(2)O(2) for 1 h to maximize ICAM-1 expression, and this was followed by treatment with actinomycin D. Total RNA was isolated at 0.5, 1, 1.5, and 2 h after actinomycin D, and steady-state levels of ICAM-1 mRNA were analyzed by Northern blotting (Fig.2). The H(2)O(2)-induced mRNA level returned to baseline level at 0.5 h (lane 3) and remained at this level up to 2 h (lanes 4-6). Both actinomycin D experiments indicated that H(2)O(2) increased the synthesis of ICAM-1 mRNA.

H(2)OIncreases Rate of ICAM-1 Gene Transcription

Fig.3compares the transcription rate of ICAM-1 with that of E-selectin, ribosomal RNA (rRNA), and glyceraldehyde-3-phosphate dehydrogenase as determined by nuclear run-on analysis. The transcription rates of E-selectin, rRNA, and glyceraldehyde-3-phosphate dehydrogenase were unaffected by H(2)O(2) over the 2-h time course. In contrast, H(2)O(2) increased the rate of ICAM-1 transcription at 1 h, and the rate remained high at 2 h, a finding that correlates with the H(2)O(2) induction of ICAM-1 message (Lo et al., 1993). These results indicate that H(2)O(2) activates ICAM-1 gene transcription.


Figure 3: Nuclear run-on analysis of H(2)O(2)-induced ICAM-1 mRNA. Slot-blot analysis of the ICAM-1 RNA transcription rates of nuclei isolated from control HUVEC and HUVEC treated with H(2)O(2) (100 µM) for 1 and 2 h (slot 3). Labeled RNA isolated from the nuclei was hybridized to immobilized DNA as indicated. For comparison, E-selectin (slot 2), glyceraldehyde-3-phosphate dehydrogenase (slot 4), and ribosomal RNA (slot 1) were also analyzed (representative of 4 separate experiments).



Antisense Oligonucleotide Prevents H(2)O-induced ICAM-1 mRNA Expression

We transfected a complementary ICAM-1 oligonucleotide that targets the 5` end of the ICAM-1 mRNA (Fig.4), to determine whether H(2)O(2)-induced ICAM-1 mRNA expression was sensitive to antisense deoxyoligonucleotides. The transfected HUVEC were exposed for 1 h with 100 µM H(2)O(2) and analyzed by Northern blot for ICAM-1 mRNA expression. Lipofection with the antisense oligonucleotide produced a concentration-dependent reduction in the ICAM-1 message (Fig.4, lanes 3 and 5). Neither control (nonsense oligonucleotide used as a negative control in lanes 4 and 6 or lipofection alone in lane 1) affected ICAM-1 mRNA expression. These data indicated that antisense oligonucleotides targeted to the ICAM-1 mRNA prevented the H(2)O(2)-induced ICAM-1 transcription.


Figure 4: Effect of an antisense oligonucleotide on the expression of ICAM-1 message induced by H(2)O(2) (100 µM) for 1 h. Total RNA was isolated from cells incubated with 50 (lane 3) or 100 µM (lane 5) antisense oligonucleotide (AS) (see ``Experimental Procedures'' for sequence of the oligonucleotide). For specificity, the effect of a nonsense oligonucleotide (NS) was assessed (lanes 4 and 6). Control RNA was isolated from cells incubated with Lipofectin alone (lane 1). A, autoradiogram of the Northern blot; B, bar graph presenting relative intensities of the ICAM-1 mRNA signal (representative of 4 separate experiments).



Antagonists of H(2)O-induced ICAM-1 mRNA Expression

We used three agents to investigate possible mechanisms underlying the induction of ICAM-1. These agents were selected to study the DNA binding proteins AP-1 (Jun/Fos) and NF-kappaB, transcription factors known to be modulated by redox mechanisms (Abate et al., 1990; Meyer et al., 1993). We used 3-aminobenzamide (3-AB), an inhibitor of poly(ADP-ribosyl)ation, to target AP-1 since 3-AB inhibited oxidant-induced c-fos expression and AP-1 binding activity (Amstad et al., 1992). The anti-oxidant pyrrolidine dithiocarbamate (PDTC) was used to target NF-kappaB since PDTC inhibited oxidant-induced NF-kappaB activity without affecting AP-1 binding activity (Schreck et al., 1992). N-Acetylcysteine (N-Cys(Ac)), a general antioxidant and precursor of glutathione (Toledano and Leonard, 1991; Abate et al., 1990), was used to alter the redox state of cells. As shown in Fig.5, 3-AB abrogated the induction of ICAM-1 message (lane 3), whereas PDTC had no effect (lane 4) suggesting a role for AP-1 in the induction of endothelial ICAM-1 transcription by H(2)O(2). Pretreatment of endothelial cells for 1 h with N-Cys(Ac) also prevented the H(2)O(2)-induced mRNA expression (lane 5).


Figure 5: Effect of 3-aminobenzamide (3AB), pyrrolidine dithiocarbamate (PDTC), and N-acetylcysteine (NAC) on H(2)O(2)-induced ICAM-1 message. Confluent HUVEC were pretreated with 3-AB (lane 3), PDTC (lane 4), or N-Cys(Ac) (lane 5) for 1 h as described under ``Experimental Procedures'' followed by exposure to 100 µM H(2)O(2) for 1 h in the presence of inhibitor. Control RNA (lane 1) and RNA from TNFalpha (100 units/ml)-treated cells (lane 6) were also analyzed. A, autoradiogram of the Northern blot; B, bar graph presenting relative intensities of the ICAM-1 mRNA signals (representative of 3 separate experiments).



H(2)OStimulates AP-1 DNA Binding Activity in Endothelial Cells

We prepared nuclear protein extracts from endothelial cells treated with H(2)O(2) for 1 h and examined DNA binding by electrophoretic mobility shift assay to study the effects of H(2)O(2) on AP-1 and NF-kappaB binding activities (Fig.6). H(2)O(2) stimulated DNA binding activity on AP-1-like binding sites of the ICAM-1 promoter (lanes 1-9), but this was not the case with NF-kappaB binding activity (lanes 10-12) consistent with the inhibitor studies above. In contrast, TNFalpha increased the binding activity on both the AP-1-like and NF-kappaB-like sequences.


Figure 6: H(2)O(2) induces AP-1 but not NF-kappaB binding activity in endothelial cells. Nuclear protein extracts of HUVEC exposed for 1 h to 100 µM H(2)O(2) or TNFalpha (100 units/ml) were incubated with TRE (lanes 1-3), AP-1/Ets (lanes 4-6), AP-1 (lanes 7-9), or NF-kappaB (lanes 10-12) binding site oligonucleotides of the ICAM-1 promoter (see ``Experimental Procedures'' for oligonucleotide sequences). Gel shift complexes indicated by the arrow were resolved by electrophoresis and DNA binding activity assessed by autoradiography.



H(2)OIncreases ICAM-1 Promoter Activity

We determined the ability of an ICAM-1 promoter construct to respond to H(2)O(2) activation signals in transient transfection assays. Fig.7A shows the structure of the ICAM-1 promoter construct and the positions of DNA binding sites of several inducible transcription factors that were identified by visually scanning the promoter. The full-length wild type construct containing nearly 1.4 kb of the ICAM-1 promoter linked to a luciferase reporter gene (ICAM-1 LUC) (Voraberger et al., 1991) was transfected into HUVEC. Fig.7B shows that the ICAM-1 promoter was maximally activated by 100 µM H(2)O(2) in HUVEC, which was in agreement with ICAM-1 mRNA expression. H(2)O(2) also induced ICAM-1 promoter activity in EAhy926 cells in a concentration-dependent manner (Fig.7C). These results indicated that H(2)O(2) increases gene transcription through activation of the ICAM-1 promoter.

H(2)OActivates Distinct Regions of ICAM-1 Promoter

We examined a nested set of 5` deletion mutation constructs containing different lengths of the ICAM-1 promoter to identify the DNA sequences within the ICAM-1 promoter required for the H(2)O(2) response. Fig.8shows that deletion of ICAM-1 promoter sequences 769 bp upstream of the ATG start codon (construct B) abrogated H(2)O(2)-induced ICAM-1 transcription. In contrast, the construct containing promoter sequences up to 981 bp upstream of the gene (Fig.8, construct A) was as responsive to H(2)O(2) as the full-length ICAM-1 promoter (-1393 ICAM-1 construct) even though this deletion construct (A) lacked an AP-1-like binding site. Therefore, DNA sequences between nucleotides 981 and 769 upstream of the gene are required for H(2)O(2)-induced ICAM-1 transcription. Within this 212-bp DNA segment are two identical 16-bp repeats, each containing binding sites for the transcription factors AP-1 and Ets (solid rectangles).

Unlike H(2)O(2), the TNFalpha response decreased only slightly (about 2-fold) with increasing deletion of the promoter to nucleotide position -769, indicating that these promoter sequences containing AP-1 binding sites, although contributing to a maximal TNFalpha response, are not essential for TNFalpha-mediated ICAM-1 transcription. The distal NF-kappaB binding site was also not essential since deletion of sequences containing this element (Fig.8, construct C) had little effect on the TNFalpha response. Indeed, a significant TNFalpha response of at least 3-fold persisted until sequences between -393 (construct D) and -176 (construct E) containing adjacent NF-kappaB and C/EBP binding sites were removed. This result is consistent with the findings of Hou et al.(1994) demonstrating cooperativity between the proximal NF-kappaB and C/EBP binding sites for the TNFalpha response in endothelial and epithelial cells.

AP-1/Ets Composite Sites Are H(2)OResponse Elements

The DNA binding studies coupled with the functional analysis of the ICAM-1 promoter suggest that the AP-1/Ets binding site repeats might be oxidant response elements. AP-1/Ets composite sites are present in a number of viral and cellular promoters including the macrophage scavenger receptor (Wu et al., 1994). AP-1/Ets elements are also known as Ras responsive elements (Westwick et al., 1994), since they can mediate activation signals transduced through the GTP-binding protein Ras, an early signaling intermediate of the AP-1 signal transduction pathway. We transfected a heterologous promoter (3 AP-1/Ets-LUC) containing three copies of the AP-1/Ets element from the macrophage scavenger receptor (MSR) gene linked to a minimal prolactin promoter-luciferase reporter gene (Wu et al., 1994) to determine whether AP-1/Ets composite elements similar to the ICAM-1 repeats could function as H(2)O(2) response elements. As shown in Table1, the MSR AP-1/Ets element is nearly identical with the ICAM-1 AP-1/Ets repeats and is also similar to anti-oxidant response elements (ARE) present in oxiprotective enzyme genes. As shown in Fig.9, AP-1/Ets-directed promoter activity increased when the transfected cells were exposed to either H(2)O(2) or TNFalpha. However, mutation of either the AP-1 or Ets binding sites prevented these responses, indicating that the AP-1 and Ets binding sites are essential for H(2)O(2)-induced promoter activity. These data define AP-1/Ets composite sites as oxidant response elements and indicate that the AP-1 and Ets binding sites functionally cooperate to activate H(2)O(2)-mediated transcription.




Figure 9: AP-1 and Ets binding sites functionally cooperate to form H(2)O(2) responsive elements. Three copies of wild type or mutant AP-1/Ets element from the macrophage scavenger receptor gene (Wu et al., 1994) linked to a prolactin TATA box luciferase construct were transfected into EAhy926 cells together with a beta-actin-beta-galactosidase expression plasmid (internal control) as described under ``Experimental Procedures.'' EAhy926 cells were exposed to H(2)O(2) (400 µM), TNFalpha (100 units/ml), or medium (control) for 24 h, and cell extracts were assessed for luciferase activity. The wild type AP-1/Ets composite element is double underlined. Mutations in either the AP-1 or Ets binding sites are delineated by a single underline. Results are expressed as mean fold increase (n = 3) of luciferase activity normalized to the beta-galactosidase activity ± S.D.



Effect of Mutations of AP-1/Ets Repeat on H(2)O(2)-induced DNA Binding Activity

We characterized the DNA binding activity on the AP-1/Ets repeats to determine whether redox-sensitive DNA binding proteins complex with these elements. Gel shift analysis revealed the formation of two specific gel shift complexes that competed with the AP-1/Ets repeat (Fig.10, lanes 1 and 5). Like TNFalpha (lane 4), H(2)O(2) increased the binding activity of these complexes (lane 2), whereas in the presence of N-Cys(Ac) H(2)O(2) did not stimulate their binding activity (lane 3). These data indicate that H(2)O(2) stimulated redox-sensitive AP-1/Ets binding activity of nuclear extracts.


Figure 10: Characterization of the H(2)O(2)-induced binding activity on the AP-1/Ets repeat. AP-1/Ets repeat oligonucleotide or oligonucleotides with mutation of either the AP-1 (AP-1 m/Ets) or Ets (AP-1/Ets-m) binding site were incubated with nuclear extracts of EAhy926 cells treated with TNFalpha (100 units/ml) or H(2)O(2) (400 µM) in the presence or absence of N-Cys(Ac) (30 mM) as indicated above each lane. The proteinbulletDNA complexes were resolved by gel electrophoresis and detected by autoradiography. Binding specificity was assessed by 50 ng of unlabeled homologous competitor oligonucleotide.



We introduced point mutations into the AP-1/Ets repeat to assess the importance of the AP-1 and Ets binding sites in the induction of these complexes. In the absence of H(2)O(2), the mutation of either the AP-1 or Ets binding sites had no effect on the constitutive binding activity (Fig.10, lanes 6 and 10). However, in the H(2)O(2)-treated cell extracts these mutations in either the AP-1 or Ets binding sites prevented the H(2)O(2)-induced binding activity (lanes 7 and 11), indicating that an intact AP-1/Ets repeat was essential for the H(2)O(2)-mediated binding activity. These muations also abrogated the TNFalpha-induced binding activity (Fig.10, lanes 8 and 12). These data suggest that the AP-1 and Ets binding sites cooperated to form redox sensitive gel shift complexes on the AP-1/Ets repeats.


DISCUSSION

In the present study, we examined mechanisms of H(2)O(2)-mediated induction of ICAM-1 mRNA expression in endothelial cells. The level of expression induced by H(2)O(2) was consistently 2- to 3-fold greater than basal ICAM-1 expression. This effect was detectable at 0.5 h, peaked at 1 h, and was sustained for at least 2 h. H(2)O(2) did not activate the transcription of E-selectin, and it has not been shown to increase expression of vascular cell adhesion molecule 1 (Bradely et al., 1993). Deletional analysis of ICAM-1 promoter sequences identified a 212-bp region required for the H(2)O(2)-mediated activation of ICAM-1 transcription. Although this region from -981 to -769 (relative to the start of translation) contributed to the TNFalpha response, it was not essential for activation of ICAM-1 transcription by TNFalpha. The major TNFalpha responsive region was localized to promoter sequences more than 300 bp downstream between -393 and -176, binding sites for C/EBP and NF-kappaB.

Within the H(2)O(2) responsive region of the ICAM-1 promoter, we identified two 16-bp repeats located 865 and 940 bp upstream of the coding region. These repeats are binding sites for the inducible transcription factors AP-1 (composed of Jun and Fos protein dimers) and Ets. No other known binding sites for nuclear regulatory factors were apparent within this H(2)O(2) responsive region. An oligonucleotide of the AP-1/Ets repeats formed H(2)O(2)-induced gel shift complexes that were sensitive to mutation of either the AP-1 or Ets binding sites, suggesting these elements mediated the H(2)O(2)-induced transcription of the ICAM-1 gene.

Similar AP-1/Ets composite elements have been found in the promoters of other genes including the macrophage scavenger receptor (MSR) gene (Wu et al., 1994). We demonstrated that the AP-1/Ets elements from the MSR gene were sufficient to induce transcription in response to H(2)O(2). Both the AP-1 and Ets binding sites were essential since mutation of these sequences reduced the induced response to H(2)O(2) suggesting these two binding sites functionally cooperated to form the H(2)O(2) response element. Wu et al.(1994) have shown that the AP-1/Ets composite elements form ternary complexes containing c-Jun, JunB, and Ets-2, which cooperate to mediate the response to phorbol ester. We also found that the AP-1/Ets elements functionally cooperated to mediate responses to TNFalpha, suggesting that H(2)O(2) and TNFalpha activate a similar set of transcription factors. However, in the context of the ICAM-1 promoter, the AP-1/Ets repeats mediated primarily the H(2)O(2) response, indicating that the AP-1/Ets elements are not necessary for the TNFalpha response even though TNFalpha has been shown to activate transcription through oxidant-mediated signals (Meyer et al., 1993) and through AP-1 binding sites (Brenner et al., 1989). Indeed, we found that N-Cys(Ac) could prevent the TNFalpha-induced binding to the AP-1/Ets elements. (^2)

The AP-1 binding sites of the AP-1/Ets repeats are also similar to the anti-oxidant response element (ARE) (Rushmore et al., 1991), a cis-acting sequence element identified in oxi-protective enzyme genes, glutathione S-transferase Ya subunit (GST Ya) and NAD(P)H:quinone oxidoreductase (Li and Jaiswal, 1992; Nguyen and Pickett, 1992; Pinkus et al., 1995). Several studies have shown that H(2)O(2) activates ARE sequences (Friling et al., 1992; Choi and Moore, 1993; Li and Jaiswal, 1994). Sequence comparisons of mammalian ARE and the ICAM-1 repeats revealed similarities between the AP-1/Ets repeats and the human NAD(P)H:quinone oxidoreductase and mouse GST Ya ARE (Table1). The mouse GST Ya contains two functional ARE sequences, one of which cooperates with an adjacent inverse Ets binding site to activate redox responses via the promoter (Bergelson and Daniel, 1994). The ICAM-1 promoter may utilize a similar mechanism to respond to H(2)O(2) since mutations in either the AP-1 or Ets binding sites abrogated the H(2)O(2)-induced binding activity. The family of AP-1 proteins (i.e. JunD, c-Fos, and JunB) have been shown to be involved in the activation of ARE (Bergelson et al., 1994; Nguyen et al., 1994), even though the ARE is functionally distinguishable from consensus AP-1 binding sites suggesting non-AP-1 proteins may also play a role in their activation (Nguyen et al., 1994; Wang and Williamson, 1994). We have shown that overexpression of JunB in epithelial cells stimulated the ICAM-1 promoter 5-fold suggesting the importance of AP-1 proteins in the response.^2

The agent 3-aminobenzamide (3-AB), which inhibits poly(ADP-ribosyl)ation and prevents oxidant-induced synthesis of c-Fos (Amstad et al., 1992), prevented the H(2)O(2)-induced ICAM-1 expression. In contrast, pyrrolidine dithiocarbamate (PDTC) (which prevents NF-kappaB activation (Schreck et al., 1992)) did not alter the H(2)O(2)-induced ICAM-1 message. H(2)O(2) also did not activate NF-kappaB binding activity as reported by Bradely et al.(1993), consistent with the lack of effect of PDTC on H(2)O(2)-induced ICAM-1 expression. In contrast, TNFalpha does activate NF-kappaB (Schreck et al., 1992), and TNFalpha-induced ICAM-1 has recently been shown to be under NF-kappaB control (Ledebur and Parks, 1995; Hou et al., 1994). Taken together, these data indicate that H(2)O(2)-mediated ICAM-1 transcription does not involve the activation of NF-kappaB. In the ICAM-1 promoter studies, we showed that TNFalpha activated a region between 393 and 176 bp upstream from the start codon which contained the C/EBP and NF-kappaB binding sites. Hou et al.(1994) showed that these two transcription factors cooperated to activate ICAM-1 transcription in response to TNFalpha. However, since we did not selectively block the TNFalpha response by specifically mutating the NF-kappaB and C/EBP sites, we cannot rule out the possibility that the AP-1/Ets repeats are also mediators of the TNFalpha response, nor can we rule out the possibility that the NF-kappaB and C/EBP sites contribute to the H(2)O(2) response. Although H(2)O(2) and TNFalpha apparently function through distinct cis-regulatory elements to activate transcription, additional studies will be required to further elucidate the specific roles these elements play in the complex regulation of the ICAM-1 gene.

Since the regulation of H(2)O(2)-induced ICAM-1 expression appears to be the result of the redox activity of H(2)O(2), we determined the effects of N-acetylcysteine (N-Cys(Ac)), an anti-oxidant that increases intracellular glutathione levels (Meyer et al., 1993). The results showed that N-Cys(Ac) inhibited AP-1/Ets binding activity and the induction of ICAM-1 expression, consistent with the findings that glutathione regulates AP-1 activity (Bergelson et al., 1994). However, the mechanism by which H(2)O(2) activation is transmitted to the ICAM-1 promoter is yet unknown. H(2)O(2) can activate the AP-1 signal transduction pathway in T-cells through tyrosine phosphorylation of kinase intermediates (Nakamura et al., 1993) raising the possibility that H(2)O(2) stimulates the ICAM-1 gene by a similar signal transduction mechanism. H(2)O(2) has also been shown to increase endothelial permeability via a protein kinase C-dependent mechanism (Siflinger-Birnboim et al., 1992). H(2)O(2) has also been shown to induce c-fos and c-jun gene expression and increase AP-1 activity (Li et al., 1994).

In summary, the present results indicate a unique mechanism of H(2)O(2)-induced activation of a cis-regulatory domain of the ICAM-1 promoter. This region situated between -981 and -769 (relative to the start codon) contains two 16-bp repeats similar to a functional AP-1/Ets composite binding site capable of transmitting H(2)O(2) activation signals to a minimal promoter. In contrast, TNFalpha activated ICAM-1 transcription through a domain between -393 and -176 containing the C/EBP and NF-kappaB binding sites of the promoter. These results indicate that H(2)O(2) and TNFalpha may mediate ICAM-1 expression by distinct intracellular mechanisms involving unique sequence elements within the promoter region of the gene.


FOOTNOTES

*
This work was supported by a grant-in-aid from the American Heart Association, American Cancer Society Grants IRG-195 and 94-10, and National Institutes of Health Grants HL27016, HL46350, and HL45638. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
The first and second authors contributed equally to this work.

To whom correspondence and reprint requests should be addressed: Dept. of Immunology/Microbiology and Pharmacology, Rush Medical College, 1750 W. Harrison St., Chicago, IL 60612. Tel.: 312-942-6259; Fax: 312-942-2808.

^1
The abbreviations used are: PMN, polymorphonuclear leukocytes; ICAM-1, intercellular adhesion molecule 1; LUC, luciferase; HUVEC, human umbilical vein endothelial cell; TNFalpha, tumor necrosis factor-alpha; N-Cys(Ac), N-acetylcysteine; PDTC, pyrrolidine dithiocarbamate; 3-AB, 3-aminobenzamide; AP-1, activator protein-1; NF-kappaB, nuclear factor kappaB; ARE, anti-oxidant response element; MSR, macrophage scavenger receptor; bp, base pair(s); kb, kilobase(s); DMEM, Dulbecco's modified Eagle's medium; MOPS, 4-morpholinepropanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; DTT, dithiothreitol; GST, glutathione S-transferase.

^2
K. A. Roebuck, unpublished result.


ACKNOWLEDGEMENTS

We thank Chris Glass for plasmids and Alison Finnegan for critical reading of the manuscript.


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