©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcriptional Regulation of the Intercellular Adhesion Molecule-1 Gene by Inflammatory Cytokines in Human Endothelial Cells
ESSENTIAL ROLES OF A VARIANT NF-kappaB SITE AND p65 HOMODIMERS (*)

(Received for publication, September 26, 1994; and in revised form, November 1, 1994)

Harry C. Ledebur (§) Thomas P. Parks (¶)

From the Department of Inflammatory Diseases, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Intercellular adhesion molecule-1 (ICAM-1) is greatly up-regulated on endothelial cells at sites of inflammation and is involved in leukocyte attachment and extravasation. Previously, we had shown that the ICAM-1 gene expression in human umbilical vein endothelial cells (HUVECs) was transcriptionally regulated by tumor necrosis factor-alpha (TNF-alpha) (Wertheimer, S. J., Myers, C. L., Wallace, R. W., and Parks, T. P.(1992) J. Biol. Chem. 267, 12030-12035). In the present investigation, TNF-alphainduced transcription was found to be initiated exclusively at two sites, 40 and 41 base pairs upstream of the translation start site. Deletion analysis of the 5` regulatory region of the ICAM-1 gene revealed a 92-base pair sequence which was both necessary and sufficient to confer TNF-alpha responsiveness to a linked luciferase reporter gene in transient transfection assays. This TNF-alpha-responsive region contained a variant NF-kappaB site at -187 to -178, which when mutated, completely abolished ICAM-1 promoter activation by TNF-alpha, interleukin-1beta, and lipopolysaccharide. Two inducible nuclear protein complexes bound to the ICAM-1 kappaB site and were identified as the NF-kappaB p65 homodimer and p65/p50 heterodimer. Overexpression of p65, but not p50, transactivated the ICAM-1 promoter in a kappaB site-dependent manner in HUVECs. In addition, p65-mediated transactivation was suppressed by co-expression of p50. Our results suggest that cytokine activation of the ICAM-1 promoter in HUVECs may critically depend on p65 homodimers binding to a variant kappaB site.


INTRODUCTION

Intercellular adhesion molecule-1 (ICAM-1) (^1)is an inducible cell surface glycoprotein belonging to the immunoglobulin su-pergene family(1, 2) . By serving as a counter-receptor for the leukocyte beta(2) integrins, LFA-1 and Mac-1, ICAM-1 participates in a wide range of inflammatory and immune responses(3, 4) . The expression of ICAM-1 on activated endothelium, for example, plays a key role in the recruitment and extravasation of circulating leukocytes at sites of tissue injury or infection(3) . ICAM-1 is expressed basally at low levels on vascular endothelium and lymphocytes, and at moderate levels on monocytes (5, 6, 7) , but can be induced to high levels on a number of cell types by stimulation with bacterial lipopolysaccaride (LPS), phorbol esters, or inflammatory cytokines, such as TNF-alpha, IL-1beta, and IFN-. Up-regulation of ICAM-1 expression by these mediators has been reported on endothelial cells(5, 6, 7, 8, 9) , keratinocytes(5) , synovial cells(10) , epithelial cells(11) , fibroblasts(5) , hepatocytes(12) , myocytes (13) , as well as activated lymphocytes(5) .

The induction of ICAM-1 cell surface expression requires de novo mRNA and protein synthesis(5, 14) . Following treatment with TNF-alpha, ICAM-1 expression on human umbilical vein endothelial cells (HUVECs) can be detected within several hours, reaches maximal levels at 8-10 h, and remains elevated for at least 72 h in the presence of cytokine(6, 7, 9) . ICAM-1 message levels are elevated within 30 min following TNF-alpha stimulation and peak at 2 h(14) . Unlike cell surface expression, ICAM-1 mRNA returns to basal levels within 24 h. Nuclear run-on assays demonstrated that TNF-alpha transiently activated ICAM-1 transcription which preceded and paralleled the changes in steady state mRNA levels. Therefore, regulation of ICAM-1 gene expression by TNF-alpha in HUVECs is controlled primarily at the transcriptional level.

The molecular events underlying the transcriptional activation of the ICAM-1 gene in response to cytokine stimulation are poorly understood. Several transcription start sites for the ICAM-1 gene have been reported, and two TATA boxes identified, but their use was dependent on the cell type and stimulus studied(15, 16, 17) . The 5`-flanking region of the ICAM-1 gene has been cloned and shown to possess potent constitutive and inducible promoter activity when linked to a luciferase or CAT reporter gene and transfected into a variety of primary cell cultures or cell lines(15, 16, 17, 18) . Sequence analysis of the 5`-flanking sequence has revealed numerous potential regulatory elements which could be involved in the cytokine activation of the ICAM-1 promoter. Recent reports have begun to identify tissue-specific and cytokine responsive regions within the ICAM-1 promoter. Look et al.(19) identified a responsive element present in the ICAM-1 5`-flanking sequence, -116 to -106 relative to the start of translation, that allowed the promoter to respond to IFN- in primary epithelial cells. Another group of investigators identified four regions of the ICAM-1 promoter which conferred PMA responsiveness in human embryonal kidney cells(20) . One of these regions, -267 to -215 relative to the start of translation, also appeared to be involved in TNF-alpha responsiveness, and mediated dexamethasone repression of both the PMA and TNF-alpha responses.

Cytokine activation of the promoters of two other inducible adhesion proteins, E-selectin and VCAM-1, in endothelial cells has been shown to critically rely on the transcription factor NF-kappaB(21, 22, 23, 24, 25, 26) . Six potential NF-kappaB sites have been identified in the ICAM-1 5`-flanking sequence and first intron, but which, if any, are involved in cytokine activation of the ICAM-1 promoter in endothelial cells has yet to be firmly established(15, 16, 17, 20, 27) . The NF-kappaB/rel family of transcription factors consists of five proteins, p65 (relA), p50 (NFKB-1), c-Rel (c-rel), Rel B (relB), and p52 (NFKB-2), which are related to each other through a N-terminal stretch of 300 amino acids called the rel homology domain(28) . DNA binding is achieved through dimerization of the family members, resulting in numerous homo- and heterodimeric combinations of NF-kappaB/rel proteins. In most cells, NF-kappaB/rel dimers are retained in the cytoplasm in an inactive state by binding to one of several inhibitor molecules which belong to the IkappaB family(29) . Activation of NF-kappaB involves the release of IkappaB and subsequent translocation of NF-kappaB/rel dimers to the nucleus. Once NF-kappaB has entered the nucleus it is capable of binding to target DNA sequences and activating transcription. The C-terminal regions of p65, c-Rel, and RelB harbor transcriptional activation domains and the in vivo transcriptional activity is attributed to p65-, c-Rel-, and RelB-containing dimers(30, 31, 32, 33, 34, 35) . p50 homodimers have been shown to transactivate in vitro, but there is no in vivo evidence for this phenomenon(36) . Not all NF-kappaB/rel dimers can bind to the same NF-kappaB sequence. It appears that variations of the NF-kappaB consensus sequence can exclude or recruit different homo- or heterodimeric combinations of NF-kappaB/rel to the site(28, 37, 38) .

The present investigation was conducted to define the molecular mechanisms involved in cytokine-induced transcription of the ICAM-1 gene in vascular endothelium. Using primary cultures of human endothelial cells as our model system, we sought to determine 1) the transcription start site(s) of the ICAM-1 gene, 2) the cis-elements within the ICAM-1 5`-flanking sequence involved in transcriptional activation of the gene, 3) the transcription factors that bind to these cis-elements, and 4) the functional relevance of these transcription factors for ICAM-1 promoter activation.


MATERIALS AND METHODS

Bacterial Strains and Plasmids

The plasmids pBHluc1.3 and p5`Delta981 were obtained from Dr. Christian Stratowa (Bender & Co., Vienna, Austria). pBHluc1.3 and p5`Delta981 contain 1344 and 932 base pairs of the human ICAM-1 upstream region, respectively (pBHluc1.3: -1393 to -49 relative to the start of translation/p5`Delta981: -981 to -49 relative to the start of translation) fused to a luciferase reporter gene. The luciferase reporter vectors pGL2 Basic and pGL2 Prom were obtained from Promega (Madison, WI). pRSV-lacZ was created by inserting the lacZ gene from pSV-beta-galactosidase (Promega) into Rc/RSV (Invitrogen, San Diego, CA). pGEMJJM, pGEM3Z+ with an altered multiple cloning region lacking a SmaI site, was provided by Dr. John Miglietta (Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT). The expression vectors pRc/CMV p65, pRc/CMV p50, and pRc/CMV Mad3 were obtained from Dr. Patrick Baeuerle (Biochemistry Institute, Albert-Ludwigs University, Freiburg, Germany).

Cell Culture

HUVECs were obtained from Cell Systems (Kirkland, WA) and maintained as described previously(9) . The cells were grown on Falcon 150-mm Petri plates (Becton Dickinson, Bedford, MA) for RNA isolation and nuclear extract preparations, and on Falcon Primaria six-well plates for transient transfection experiments.

RNA and DNA Biochemistry

Large scale DNA plasmid preparations were isolated from CsCl gradients after Nonidet P-40 detergent lysis(39) . Small scale plasmid preparations were carried out using the Plasmid Mini Kit (Qiagen, Chatsworth, CA). Total RNA was isolated from 2-4 times 10^7 HUVECs using the Phase Lock Gel System from 5` 3`, Inc. (Boulder, CO). DNA sequencing was performed using a Taq DyeDeoxy Termination Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and a Perkin-Elmer Thermal Cycler (model 480). The sequencing reactions were analyzed on an Applied Biosystems 373A DNA sequencer.

Primer Extension Analysis

Primer extension analyses were performed as described elsewhere (40) . Oligonucleotides were synthesized on an Applied Biosystems model 392 DNA/RNA synthesizer. The 5` ends of the oligonucleotides listed below were labeled with [-P]ATP and T4 polynucleotide kinase (Life Technologies, Inc., Gaithersburg, MD) and used to determine the 5` end(s) of the human ICAM-1 transcript: PE1 (5`-TGCTGCTTTCCCGGAAACCTCGTGCCTTCCCCTCCGGAAC-3`, -100 to -139 relative to the translation initiation site), PE2 (5`-ACACAGATGTCTGGGCATTGCCAGGTCCTGGGAACAGAGC-3`, +55 to +94), PE3 (5`-GGTGCCCGGGCCGAGAGGGTCATCCTCCCTCGCTGGCCGC-3`, -169 to -208), PE4 (5`-GCCGCTTCAGCTCCGGAATTTCCAAGCTAAAGCAATCGGG-3`, -206 to -243). The mobility of the extended primers in a denaturing polyacrylamide gel was compared to that of a S-dATP dideoxy ladder of known sequence, M13mp19. The sequence ladder was generated using a primer (5`-CGCCAGGGTTTTCCCAGTCACGAC-3`) whose 3` end was 71 bp from the HindIII site of M13mp19.

Construction of Reporter Plasmids

A series of deletions were created through the 5`-flanking sequence of the ICAM-1 gene by restriction digestion of the parent plasmid pBHluc1.3. Deletion fragments of the upstream region were initially subcloned into pUC18 in order to facilitate cloning into the luciferase reporter vector pGL2 Basic. The following 5` deletion constructs were generated.

pGL 1.3

The KpnI-SalI fragment of pBHluc1.3 containing 1344 base pairs of the ICAM-1 upstream region (-1353 to -9 relative to the start of transcription) was cloned into the luciferase reporter vector pGL2 Basic that had been digested with KpnI and XhoI.

pGL.98R1

932 base pairs of the ICAM-1 upstream region (-941 to -9 relative to the start of transcription) was removed from the plasmid p5`Delta-981 by digesting with EcoRI, blunted by treatment with Klenow fragment, and then cut with SalI. This fragment was subcloned into pUC18 that had been cleaved with SmaI and SalI. The resulting plasmid was then digested with KpnI and SalI to liberate the 932-base pair ICAM-1 fragment which was subsequently cloned into pGL2 Basic digested with KpnI and XhoI.

pGL XhoI, pGL StuI, and pGL DraI

pGL 1.3 was digested with either XhoI, StuI, or DraI (with the XhoI overhang blunted with Klenow) and SalI to generate 565 (-574 to -9 relative to the start of transcription), 436 (-445 to -9), and 313 (-322 to -9)-base pair fragments of the ICAM upstream region, respectively. These fragments were each cloned into pUC18 digested with SmaI and SalI, removed from the resulting plasmids as KpnI/SalI fragments, and cloned into pGL2 Basic as before.

pGL H3 and pGL PstI

pGL 1.3 was digested with either HindIII or PstI and blunted with Klenow fragment or T4 polymerase, respectively. These fragments were subcloned into pUC18 linearized with SmaI and orientation of the resulting plasmids determined by digestion with SalI. Fragments of the ICAM-1 upstream region (pGL H3, -277 to -9; pGL PstI, -105 to -9) were cloned into pGL2 Basic as KpnI-SalI fragments as above.

pGL SmaIA

pGL StuI was digested with SmaI. The resulting vector and a 92-base pair fragment (-227 to -136 of the ICAM-1 upstream region) were isolated and ligated, thereby eliminating the -445 to -228 region of pGL StuI. Constructs with the correct orientation (determined by sequencing) contained the -227 to -9 fragment of the ICAM-1 upstream region inserted into pGL2 Basic.

pGL SmaIB

pGL StuI was digested with SmaI, gel-purified, and religated to eliminate the -445 to -136 region of pGL StuI, leaving the -135 to -9 region of the ICAM-1 promoter intact.

pGL 1.3SmaDelta

The HindIII fragment of pGL 1.3 was cloned into pGEMJJM. The resultant plasmid (pGEMJJM1.3H3) was digested with SmaI and religated back to itself after the removal of the -227 to -136 region of the ICAM-1 upstream region. The HindIII fragment was removed from this vector (pGEMJJM1.3H3Delta) and cloned back into pGL 1.3 recreating the original reporter construct with the -227 to -136 region deleted.

pGL2 Prom SASB

The SmaI fragment of pGEMJJM1.3H3 containing the -227 to -136 region of the ICAM-1 promoter was cloned into the SmaI site of pGL2 Prom, upstream of the SV40 early promoter, and orientation determined by sequencing.

pGL2 Prom 38bpwt

A synthetic double-stranded oligonucleotide containing the -200 to -163 region of the ICAM-1 promoter flanked by 5` KpnI and 3` BglII sites was directly cloned into pGL2 Prom.

pGL2 Prom 38bpkappaB

pGL2 Prom 38bpkappaB was identical to pGL2 Prom 38bpwt except the ICAM-1 sequence between -187 and -178 was changed from TGGAAATTCC to TctAgATTag. All pGL2 Basic and pGL2 Prom constructs containing fragments of the ICAM-1 upstream region were verified by sequence analysis using primers flanking the 5` and 3` boundaries of the inserts, GLprimer 1 and GLprimer 2 (Promega).

Site-directed Mutagenesis

Site-directed mutagenesis was performed using the Altered Sites Mutagenesis System (Promega). Briefly, the HindIII fragment of pGL 1.3 was subcloned into the supplied mutagenesis vector pAlter-1 to create pAlter1.3H3. The resulting plasmid was subjected to mutagenesis using the following oligonucleotides: MP8 (5`-GCCGCTTCAGCTCCCTAATCTAGAAGCTAAAGCAATCG-3`) and MP9 (5`-GCCGAGAGGGTCATAGATCTTCGCTGGCCGCTTC-3`). MP8 was designed to alter the sequence at -187 to -178 (relative to the start of transcription) from TGGAAATTCC to TctAgATTag (creating an XbaI site) and MP9 the sequence at -157 to -146 from GGGAGGATGACC to aGatctATGACC (creating a BglII site). Single-stranded DNA of pAlter1.3H3 was isolated from JM109 cells after a 24-h infection with the phage R408 and mutagenized following the supplier's protocol. pAlter1.3H3 clones bearing the desired mutations were screened by restriction digestion followed by sequencing. The HinDIII fragments containing the mutations created with MP8 and MP9 were then subcloned back into pGL 1.3 linearized with HindIII to create pGL 1.3kappaB and pGL 1.3Ets, respectively. These constructs were verified by sequence analysis using three different primers: GLprimer1, GLprimer2, and MP2 (5`-GGCAGTATTTAAAAGTACTGAAGAAACCGCTTAGCGC-3`, corresponding to the sequence -332 to -295 relative to the start of transcription).

Transient Transfection of HUVECs

HUVECs (leqpassage 4) were seeded on six-well plates (4.0 times 10^5 cells/well) in EGM medium (Clonetics, San Diego, CA) and refed 24 h later with HUVEC growth medium(9) . The cells were transfected with plasmids using a modified DEAE-dextran protocol 24 h after refeeding. The plasmid of interest and pRSV-lacZ (12 µg of each) were co-precipitated with ethanol, resuspended in 100 µl of phosphate-buffered saline, without Ca2 and Mg2 (PBS) (Life Technologies, Inc.), containing 10 mM HEPES (pH 7.3), and added to 2.9 ml of PBS-HEPES containing 250 µg/ml DEAE-dextran (average M(r) of 500,000; Sigma). The cells were washed twice with PBS, and 500 µl of the DNA/DEAE-dextran solution was added to each well. The plate was incubated for 30 min at 37 °C with rocking every 5 min. Two ml of EGM medium supplemented with 80 µM chloroquine was then added to each well, and the plate incubated for an additional 2.5 h at 37 °C. The medium was removed and the cells shocked by addition of 1 ml of 10% Me(2)SO in EGM medium for 2.5 min. The Me(2)SO was removed and replaced with EGM medium. The following day, TNF-alpha (100 units/ml) was added to half of the wells, and 8 h later luciferase and beta-galactosidase activities were determined.

Luciferase and beta-Galactosidase Assays

Briefly, the cells were washed twice with PBS and 250 µl of lysis buffer (supplied in Promega's luciferase assay kit) was added to each well. After 15 min at room temperature, the lysates were transferred to microcentrifuge tubes and cell debris removed by centrifugation at 12,000 times g for 5 min. Luciferase activity was determined using 20 µl of the clarified lysate and 100 µl of luciferase assay substrate (Promega) in a Berthold AutoLumat LB953 luminometer. beta-Galactosidase assays were carried out using 70 µl of the lysates as described previously(39) . The luciferase activity of each well was first normalized to beta-galactosidase activity before calculating the induction values reported under ``Results.''

Electrophoretic Mobility Shift Assays (EMSA)

HUVEC nuclear extracts were prepared using a procedure adapted from Dignam et al.(41) . Cells were grown to confluence on 150-mm Petri plates (1 times 10^7 cells/plate), treated with TNF-alpha (100 units/ml) for 20 min, washed two times with ice-cold PBS, scraped in 5 ml of PBS/plate, and pelleted by centrifugation at 2000 rpm for 5 min at 4 °C. Cell pellets were resuspended in 40 µl of Buffer A (10 mM HEPES (pH 7.9), 1.5 mM MgCl(2), 10 mM KCl, 0.5 mM DTT, 10 µg/ml leupeptin, 0.5% Nonidet P-40)/plate, transferred to a microcentrifuge tube, and placed on ice for 10 min. The tubes were centrifuged at 12,000 times g for 10 min at 4 °C and the supernatant removed. The nuclear pellet was resuspended in 15 µl of Buffer C (20 mM HEPES (pH 7.9), 25% glycerol, 0.72 M NaCl, 1.5 mM MgCl(2), 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT, 10 µg/ml leupeptin)/plate and incubated on ice for 15 min. Samples were centrifuged for 10 min as before and the supernatant collected (nuclear extract). The nuclear extract was diluted with 60 µl of Buffer D (20 mM HEPES (pH 7.9), 20% glycerol, 50 mM KCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT, 10 µg/ml leupeptin)/plate, frozen in liquid nitrogen, and stored at -80 °C. Protein concentration was determined using a Bio-Rad protein assay kit (Bio-Rad) with bovine IgG as a standard.

DNA fragments were end-labeled with [alpha-P]dATP and dGTP, dCTP, and dTTP by incubation with Klenow fragment at 37 °C for 30 min. The labeled probes were separated from unincorporated nucleotides using STE SELECT-D G-25 spin columns from 5` 3`, Inc. Nuclear extract (10-20 µg protein) was incubated with 0.5-1 ng (30,000 cpm) of labeled probe for 30 min at room temperature in a binding buffer containing 10 mM HEPES (pH 7.5), 1 mM EDTA, 1 mM beta-mercaptoethanol, 75 mM NaCl, 0.1 mg/ml poly(dI-dC)bulletpoly(dI-dC), and 5% glycerol. Electrophoresis was carried out using 4% native acrylamide gels (50 mM Tris base (pH 8.5), 380 mM glycine, 2.5 mM EDTA, 2.5% glycerol). Gels were vacuum-dried and exposed to x-ray film overnight. In some experiments, nuclear extracts were preincubated with antibodies or unlabeled oligonucleotides to antagonize the binding reactions. Rabbit polyclonal antibodies directed against various transcription factors were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Double-stranded oligonucleotides containing transcription factor consensus binding sites were obtained from Life Technologies, Inc. (Sp1), Santa Cruz Biotechnology (consensus and mutant NF-kappaB), or synthesized and annealed by Midland Certified Reagent (Midland, TX). Recombinant p65 for EMSA was produced in vitro using the plasmid pRc/CMV p65 and the TNT T7 Coupled Wheat Germ Extract System from Promega.

UV Cross-linking of Protein-DNA Complexes

A photoreactive P-labeled DNA probe was prepared which encompassed the -200 to -163 region of the ICAM-1 upstream sequence. The 38-base template oligonucleotide 5`-GATTGCTTTAGCTTGGAAATTCCGGAGCTGAAGCGGCC-3` was annealed to a 15-base primer oligonucleotide 5`-GGCCGCTTCAGCTCC-3` and filled in using Klenow fragment in the presence of 10 µCi [alpha-P]dATP, 200 µM dCTP, 200 µM dGTP, 100 µM dTTP, and 100 µM 5-bromo-2`-deoxyuridine 5`-triphosphate (BrdUrd). EMSAs were run as described above using the BrdUrd-substituted probe, and the protein-DNA complexes cross-linked by UV irradiation for 30 min on a Foto/Prep I transilluminator (Fotodyne, Hartland, WI). The gel was vacuum-dried, and individual complexes were excised from the gel after localization by autoradiography. The gel slices were rehydrated in 50 µl of SDS-PAGE sample buffer containing protein molecular weight standards (NOVEX, Encinitas, CA), heated for 5 min at 95 °C, and subjected to discontinuous SDS-PAGE using a 5% stacking gel and a 10% resolving gel(42) . The gel was stained with Coomassie Blue and destained to visualize protein standards, vacuum-dried, and subjected to autoradiography to locate cross-linked proteins.


RESULTS

Determination of the ICAM-1 Transcription Start Site(s) in Endothelial Cells

Two start sites for transcription of the ICAM-1 gene have been reported in A549 and Hs913T cells, located 319 and 41 bases upstream from the start of translation(16) . To determine which, if any, of these start sites were used in HUVECs, primer extension analysis was carried out on total RNA isolated from unstimulated cells or cells stimulated with either TNF-alpha or PMA (Fig. 1). Proximal start site usage was investigated using an oligonucleotide primer, PE2, that extended from +94 to +55 relative to the ICAM-1 translation start site. Primer extension reactions using this oligonucleotide yielded two products 134 and 135 bases in length from RNA isolated from TNF-alpha- or PMA-stimulated cells, but not from unstimulated cells. The distal start site was investigated using the oligonucleotide PE4, extending from -204 to -243, and yielded no reverse transcribed products in any of the three conditions. If transcription began at the distal site, PE4 would have yielded a product approximately 115 bases in length (Fig. 1). Two additional oligonucleotides, PE1 (-139 to -100) and PE3 (-208 to -169), were used as primers to confirm the lack of distal start site usage and to investigate the possibility of cryptic intermediate start sites. No reverse-transcribed products were obtained using these primers (data not shown). Therefore, unlike A549 cells, ICAM-1 transcription following TNF-alpha and PMA treatment in HUVECs began at the same sites, 40 and 41 base pairs upstream from the start of translation and 24 and 25 base pairs downstream from the proximal TATA box. In this study, the guanine residue 25 base pairs downstream from the TATA box will be referred to as +1, the transcription start site.


Figure 1: Identification of the ICAM-1 gene transcription start sites. Total RNA was isolated from HUVECs following a 2-h stimulation with TNF-alpha (T; 100 units/ml), a 4-h stimulation with PMA (P; 50 ng/ml), or from unstimulated cells(-). Primer extension analysis on the RNA was performed using the ICAM-1-specific oligonucleotide primers PE2 or PE4, and subjected to electrophoresis through a denaturing polyacrylamide gel (see ``Materials and Methods''). Lengths of the extended products were determined by comparison with a sequence ladder of M13mp19; sizes in bases are indicated. Arrows mark the position of extension products on the ICAM-1 sequence from -30 to -53 (relative to the start of translation). The numbers on the top and bottom of the ICAM-1 sequence indicate the product length if extension was terminated at that residue. Similar results were obtained with three different RNA preparations.



Identification of the TNF-alpha-responsive Region of the ICAM-1 Promoter in HUVECs

To locate the cis-regulatory element(s) present within the 5`-flanking sequence of the ICAM-1 promoter that confers TNF-alpha responsiveness, we constructed a series of deletions through the 5`-flanking region of the ICAM-1 gene and fused them to a luciferase reporter gene in the vector pGL2 Basic (Fig. 2A). These reporter constructs were transiently transfected into HUVECs and the luciferase activities of control and TNF-alpha treated cells were determined. High levels of induction were observed with the reporter constructs containing ICAM-1 5`-flanking sequence from -1353 (pGL 1.3) to -277 (pGL H3). However, further deletion to -105 (pGL PstI) resulted in a complete loss of TNF-alpha-induced promoter activity. There was a reproducible decrease in the induction ratio (6.8 to 3.7) upon deletion of 412 and 779 base pairs from the 5` end of pGL 1.3 (pGL.98RI and pGL XhoI, respectively), but all subsequent deletions retained approximately a 4-fold induction in response to TNF-alpha until the -277 to -106 region was removed. Although the region between -1353 and -547 may contain elements that play an augmentative role, it was apparent that the ICAM-1 5`-flanking sequence between -277 and -106 was required for the promoter to respond to TNF-alpha. Two additional deletions were constructed within this region to further define the 5` and 3` boundaries of this TNF-alpha-responsive region, -227 (pGL SmaIA) and -135 (pGL SmaIB) (Fig. 2B). pGL SmaIA and pGL H3 had similar induction ratios in response to TNF-alpha treatment, whereas pGL SmaIB was not induced by TNF-alpha. Therefore, we concluded that the region of the ICAM-1 promoter most critical for TNF-alpha induction in endothelial cells was located between -227 and -136.


Figure 2: Identification of a critical TNF-alpha-responsive region of the ICAM-1 promoter. A series of 5` deletions (A and B) and an internal deletion (C) of the 1.3-kilobase fragment of the ICAM-1 promoter were fused to a promoterless luciferase reporter gene, or an internal fragment (-227 to -136) was placed upstream of the SV40 promoter-linked luciferase reporter gene as described under ``Materials and Methods.'' The deletion constructs were transiently transfected into HUVECs with the control plasmid, pRSV-lacZ, using DEAE-dextran. Twenty-four hours after transfection, one-half of the transfectants were stimulated with TNF-alpha (100 units/ml) for 8 h. The cells were then lysed and assayed for luciferase and beta-galactosidase activity. The luciferase activity of individual transfectants was subsequently normalized to beta-galactosidase activity. Transfections were performed in triplicate (stimulated and unstimulated) and the normalized luciferase activities averaged. Average stimulated values were divided by average unstimulated values to give the induction ratios reported. The results presented were representative of three separate experiments.



The 92-bp ICAM-1 TNF-alpha-responsive region was cloned into the luciferase reporter vector, pGL2 Prom, upstream of the SV40 early promoter (pGL2 Prom SASB). The empty promoter vector, pGL2 Prom, exhibited very low basal activity and was not responsive to TNF-alpha treatment when transfected into HUVECs. However, the 92-bp ICAM-1 TNF-alpha-responsive region conferred TNF-alpha inducibility to the SV40 promoter similar to that obtained with the shortest inducible deletion construct, pGL SmaIA (Fig. 2B). Conversely, removal of the 92-bp responsive region from pGL 1.3 (pGL 1.3SmaDelta) completely eliminated the ability of the full-length construct to respond to TNF-alpha (Fig. 2C). It appeared, then, that this 92-bp region of the ICAM-1 5`-flanking sequence was both sufficient and necessary for the ICAM-1 promoter to drive expression of a luciferase gene in response to TNF-alpha.

Localization of Protein Binding Sites within the TNF-alpharesponsive Region

Sequence analysis of the TNF-alpha-responsive region between -227 and -136 revealed potential binding sites for four transcription factors, Sp1 (-206 to -201) (43) , C/EBP (-198 to -195)(44) , ets (-153 to -150)(45) , and NF-kappaB (-187 to -178) (46) (Fig. 3). To determine which of these sequence motifs functioned as protein binding sites, the 92-bp responsive region was labeled and used as a probe for EMSA. Two retarded complexes were observed using nuclear extracts prepared from TNF-alpha treated HUVECs that were absent in nuclear extracts from untreated cells (Complexes A and B, Fig. 4A). Inclusion of excess unlabeled 92-bp fragment in the binding reaction resulted in a complete loss of the TNF-alpha-inducible complexes, indicative of specific protein-DNA interactions. In addition, a prominent faster migrating complex was observed in nuclear extracts prepared from both unstimulated and stimulated cells (Complex C, Fig. 4A). This constitutive complex was never completely eliminated by the addition of excess unlabeled 92-bp fragment. This may have been due to the presence of two different retarded complexes with similar mobilities, one being due to nonspecific binding. The EMSAs have been repeated numerous times using different extract preparations with similar results. One variation in the gel shift pattern was the occasional appearance of a complex in the nuclear extracts prepared from unstimulated cells with a mobility similar to Complex B. Whether this complex and Complex B were the same has not been resolved. The formation of inducible nuclear complexes was maximal 20 min following treatment of HUVECs with TNF-alpha (Fig. 4A). Increasing the incubation time with TNF-alpha to 45 or 90 min did not appreciably change the relative abundance of the inducible complexes (data not shown). Using double-stranded oligonucleotides containing potential transcription factor binding sites as unlabeled competitors, we indirectly identified the binding site of the inducible complexes within the 92-bp fragment (Fig. 4B). The inducible complexes A and B were eliminated with competitors containing a consensus NF-kappaB site or the ICAM-1 NF-kappaB site, whereas a competitor with a point mutation in the consensus NF-kappaB site competed poorly for the inducible complexes. The only other competitor that exhibited an ability to compete for the inducible complexes was one containing a consensus binding site for the transcription factor C/EBPbeta or NF-IL6(47) . However when this experiment was repeated using the ICAM-1 C/EBP site, no competition for the inducible or constitutive complexes was observed (data not shown). Interestingly, the same competitors that had an effect on the inducible complexes also had a slight effect on the constitutive complex, complex C, suggesting that the inducible and constitutive complexes may share similar or overlapping binding sites.


Figure 3: Sequence of the TNF-alpha-responsive region of the ICAM-1 promoter. ICAM-1 sequence from -227 to -136 relative to the start of transcription, comprising the TNF-alpha regulatory region, is shown. Potential transcription factor binding sites were identified based on sequence homologies to their published consensus sequences: Sp1 (-96 to -199), C/EBP family (opposite strand, -201 to -206), NF-kappaB (-176 to -187), and the ets family (-150 to -153).




Figure 4: Nuclear protein complexes which bind to the TNF-alpha-responsive region of the ICAM-1 promoter. Nuclear extracts were prepared from HUVECs incubated in the presence or absence of TNF-alpha (100 units/ml) for 20 min. A radiolabeled probe (-227 to -136, relative to the start of transcription) was incubated with nuclear extract (10 µg of protein) for 30 min at room temperature and protein-DNA complexes resolved by electrophoresis. A, nuclear extracts from unstimulated (lanes 1 and 2) and TNF-alpha-stimulated (lanes 3 and 4) HUVECs were analyzed for binding activity. Lanes 2 and 4 contained a 50-fold molar excess of unlabeled probe as a competitor. Specific protein-DNA complexes (A, B, and C) are indicated by arrows. B, competition EMSA using nuclear extract prepared from TNF-alpha-treated HUVECs and various unlabeled double-stranded oligonucleotides. The oligonucleotides contained portions of the ICAM-1 regulatory element identified in Fig. 3and other transcription factor binding sites. They were present in the binding reactions at a 50-fold molar excess over probe as follows: lane 1, no competitor; lane 2, ICAM-1-responsive region -227 to -136; lane 3, consensus Sp1; lane 4, consensus C/EBPbeta; lane 5, ICAM-1 NF-kappaB site -189 to -177; lane 6, consensus NF-kappaB site; lane 7, consensus NF-kappaB site with a single point mutation; lane 8, ICAM-1 promoter region -177 to -166; lane 9, ICAM-1 ets site -160 to -148.



To confirm that the inducible complexes bound to the NF-kappaB element, methylation interference footprint assays were performed. The binding site for the upper inducible complex, Complex A, was determined to be between -187 and -178, corresponding to the NF-kappaB site identified above (Fig. 5). Evident from the protection pattern was the importance of 4 guanine residues for binding of complex A to the site. In addition, the lower inducible complex, Complex B, produced a footprint identical to that obtained for Complex A (data not shown), indicating that these two distinct protein complexes recognized and bound to the same NF-kappaB site. No binding site for the constitutive complex was evident by methylation interference footprinting (data not shown).


Figure 5: Methylation interference footprinting of complex A to the ICAM-1 kappaB site. Coding and noncoding strands were individually radiolabeled and used in binding reactions with nuclear extract from TNF-alpha-treated HUVECs. Reactions were scaled up 8-fold to permit the isolation of probe bound to complex A (B) and unbound free probe (U). Isolated complexes were then cleaved with piperidine and subjected electrophoresis on a denaturing polyacrylamide gel as described in detail in (40) . ICAM-1 sequences (-196 to -169) of both the coding and noncoding strands are shown. Specific residues are marked with an asterisk to represent those residues that when methylated interfere with the binding of Complex A. The ICAM-1 kappaB site identified in Fig. 3is indicated with brackets.



Functional Significance of the ICAM-1 NF-kappaB Site

In order to determine the functional importance of the NF-kappaB site, we mutated the site by altering the guanine residues implicated in complex formation from the previous footprinting studies. The NF-kappaB site was mutated from TGGAAATTCC to TctAgATTag in the context of the full-length reporter construct pGL 1.3 (pGL 1.3kappaB) and transiently transfected into HUVECs. In addition, the ets site was mutated from GGGAGGATGACC to aGatctATGACC to serve as a control (pGL 1.3Ets). Mutation of the kappaB site rendered the full-length reporter construct completely nonresponsive to TNF-alpha treatment, whereas the ets site mutation had no effect (Fig. 6A). Separate EMSA experiments confirmed that ICAM-1 promoter fragments bearing the NF-kappaB site mutation were incapable of binding the two inducible nuclear complexes (data not shown).


Figure 6: Functional consequences of mutating the ICAM-1 kappaB site. A, the ICAM-1 kappaB and ets sites were mutated in the context of the full-length construct, pGL 1.3, as detailed under ``Materials and Methods.'' pGL 1.3, pGL 1.3kappaB, pGL 1.3 Ets, and pGL2 Basic were transiently transfected into HUVECs and the cells subsequently stimulated with TNF-alpha (100 units/ml) for 8 h. B, pGL 1.3 and pGL 1.3kappaB were transiently transfected into HUVECs and stimulated with either TNF-alpha (100 units/ml), PMA (20 ng/ml), IL-1beta (200 ng/ml), or LPS (1 µg/ml) for 8 h. The induction values given in A and B were calculated as described in the legend to Fig. 2and are representative of three separate experiments, each performed in triplicate.



The ICAM kappaB mutant construct was also used to assess the importance of this element for induction of the ICAM-1 promoter in response to other proinflammatory stimuli. In addition to TNF-alpha, pGL 1.3 responded to other proinflammatory agents known to up-regulate ICAM-1 expression in HUVECs, including IL-1beta, PMA, and LPS (Fig. 6B). It was evident that the NF-kappaB mutation eliminated the ability of the reporter construct to respond to TNF-alpha and IL-1beta. Although the mutation significantly lowered PMA induction, the reporter construct still retained a low level of PMA induction. The induction observed in response to LPS treatment was small, but when the NF-kappaB site was mutated, there was a consistent decrease to unstimulated levels. Therefore, in HUVECs, mutation of the NF-kappaB site between -187 and -178 completely eliminated the induction of the ICAM-1 promoter by TNF-alpha, IL-1beta, and LPS, and largely, but not completely, suppressed induction by PMA.

Identification of the Proteins Which Bind to the ICAM-1 NF-kappaB Site

To facilitate these studies, the 92-bp probe (-227 to -136) used in the initial gel retardation experiments was shortened to 38 bp (-200 to -163) to improve resolution of the inducible complexes and to reduce background. As previously observed for the 92-bp probe, the 38-bp probe bound two inducible complexes which were specifically competed with unlabeled 38-bp fragment (Fig. 7A). In addition, a 38-bp competitor containing the NF-kappaB site mutation described above did not affect inducible complex formation, even at a 240-fold molar excess over radiolabeled probe (Fig. 7B). A comparison of the ability of the unlabeled 38-bp fragment to compete for inducible complex formation with that of a 10-bp oligonucleotide containing only the ICAM-1 NF-kappaB site, revealed that the 10-bp competitor did not compete for the lower complex as effectively as the 38-base pair fragment, suggesting that the lower complex, or a component of the lower complex, was influenced by flanking sequence (Fig. 7B).


Figure 7: Selective disruption of inducible complex formation using anti-p65. A, nuclear extracts from unstimulated (lanes 1 and 2) and TNF-alpha-stimulated (lanes 3 and 4) HUVECs were incubated with a 38-bp radiolabeled probe (ICAM-1 -200 to -163) containing the ICAM-1 kappaB site. Lanes 2 and 4 contained a 50-fold molar excess of unlabeled probe as a competitor. The inducible complexes are indicated by arrows. B, competition of the upper and lower inducible complexes with unlabeled 38-bp fragment (lanes 2-4), a mutant ICAM-1 kappaB 38-bp fragment (containing the same kappaB site mutation as Fig. 6; lanes 5-7), and a 10-bp fragment containing only the kappaB site (-187 to -178) (lanes 8-10). The molar excess of competitor over probe is indicated above each lane. Lane 1 did not contain competitor. C, specific antisera to various members of the NF-kappaB/rel family of transcription factors were tested: p65 (lane 2), c-Rel (lane 3), RelB (lane 4), p50 (lane 5), and p52 (lane 6). As controls, lane 1 did not contain antibody and lane 7 contained c-fos antibody. D, antisera to various members of the bZIP family of transcription factors were tested: c-jun (lane 3), C/EBPalpha (lane 4), C/EBPbeta (lane 5), ATF-1 (which cross-reacts with CREB-1 and CREM-1, lane 6), ATF-2 (lane 7), ATF-3 (lane 8), and CREB-2 (which cross-reacts with ATF-4, lane 9). Lane 1 did not contain antibody and anti-p65 was included in lane 2 as a positive control.



To determine which proteins were responsible for the formation of the two inducible nuclear complexes, we initially attempted to disrupt binding using specific antisera directed against members of the NF-kappaB/rel family (p65, p50, p52, c-Rel, and RelB) (Fig. 7C). An antiserum against c-fos was included as a control. Although the addition of serum protein tended to diminish binding of the complexes in a nonspecific manner, only the antiserum directed against p65 had a dramatic effect on the complexes. It eliminated the formation of both the upper and lower complexes. None of the other NF-kappaB/rel antibodies affected the formation of inducible complexes in a reproducible manner, although p50 and c-rel proteins and p100 (p52) message have been detected in HUVECs(48, 49) . NF-kappaB/rel proteins have recently been shown to interact with other proteins, particularly members of the bZIP family of transcription factors (50, 51, 52) . In order to investigate the possibility that one of the complexes resulted from an interaction between p65 and a non-rel protein, antisera to various bZIP proteins were tested in EMSAs (Fig. 7D). None of the antibodies tested, including those directed against c-jun, C/EBPalpha, C/EBPbeta, ATF-1, ATF-2, ATF-3, and CREB-2, had any effect on inducible complex formation. From these experiments, we concluded that p65 was a component of both the upper and lower complexes, but we were unable to identify other factors that might contribute to the formation of the two complexes.

To further define the polypeptide composition of the inducible complexes, the proteins of the upper and lower complexes were UV-cross-linked to a BrdUrd-substituted 38-bp probe. For comparison, recombinant p65 (rp65) was included in the cross-linking experiments. Recombinant p65 produced a single retarded complex with a mobility similar to that of the upper complex when analyzed by EMSA (Fig. 8A), and like the upper complex, could be eliminated by addition of p65 antibody or unlabeled probe (data not shown). The polypeptides of the upper and lower complexes and rp65 were cross-linked to the probe by exposure to UV light, excised from the gel, and analyzed on a SDS-polyacrylamide gel (Fig. 8B). The upper complex and rp65 contained a single cross-linked polypeptide of approximately 75 kDa. The lower complex also contained a 75-kDa cross-linked polypeptide, as well as a smaller 59-60 kDa adduct. Based on the comparison of antibody cross-reactivity and cross-linking experiments with rp65, we concluded that the larger polypeptide present in both inducible complexes was p65. These results indicated that the upper complex was due to the binding of a p65 homodimer, and the lower complex was a heterodimer of p65 and another polypeptide approximately 15 kDa smaller than p65. Thus, the smaller polypeptide exhibited a molecular weight similar to that expected for p50.


Figure 8: UV cross-linking of the inducible complexes. A photoreactive 38-bp probe was prepared as described under ``Materials and Methods.'' A, EMSA using the photoaffinity probe and nuclear extract prepared from TNF-alpha treated HUVECs (lane 1) and recombinant p65 (lane 2). B, the upper and lower complexes were UV-cross-linked to the probe, excised from the gel, and subjected to SDS-PAGE. Molecular weights of the cross-linked products were estimated by comparison with molecular weight standards.



Since it was possible that the p50 antiserum did not react strongly with the p50/p65 heterodimer in the EMSA binding reaction, p50 or p65 was immunodepleted from nuclear extracts using peptide-specific p50 or p65 antisera conjugated to agarose beads and the depleted supernatant used in the EMSA binding reaction. Antibody specificity was demonstrated by pretreating the antiserum with the peptide to which the antiserum was raised or an irrelevant peptide (Fig. 9A). Immunodepletion of p50 specifically eliminated the lower complex, an effect not observed when the antiserum was preincubated with the p50 peptide. In contrast, the p65 peptide did not block immunodepletion of the lower complex by anti-p50. As expected, both the upper and lower complexes were eliminated when nuclear extracts were depleted of p65. This effect was reversed by pretreatment of the antiserum with p65 peptide, but not p50 peptide. Immunodepletion of other NF-kappaB/rel family members, i.e. c-rel, relB, and p52, had no effect on either of the inducible complexes (data not shown).


Figure 9: Immunochemical identification of p50 as a component of the lower complex. A, immunodepletion of the lower complex. Peptide-specific antibodies conjugated to agarose beads were used to immunodeplete p50 or p65 from nuclear extracts prepared from TNF-alpha-treated HUVECs. To demonstrate specificity, conjugated antibody (25 µl) was treated with an excess of specific peptide to which the antibody was raised, an irrelevant peptide, or PBS for 2 h at room temperature. The conjugated antibodies were pelleted by centrifugation and incubated with nuclear extract (88 µg) in the presence of the appropriate peptide or PBS (50 µl total volume) for 4 h at 4 °C. The conjugated antibodies were then collected by centrifugation and 10 µl of the immunodepleted supernatant analyzed by EMSA. The antisera and peptides used are indicated at the top of the figure. Untreated nuclear extract was included in the EMSA as an additional control (lane 4). B, immunopurification of the lower complex. Nuclear extract prepared from TNF-alpha-treated HUVECs (500 µg) was incubated with 60 µl of anti-p50 conjugated to agarose beads for 4 h at 4 °C. The conjugated antibody was collected by centrifugation and washed five times with PBS. The protein retained on the conjugated antibody was eluted using two sequential 2-h incubations with 100 µl of the p50 peptide. The elutates were combined, concentrated to a final volume of 10 µl using an Amicron Microcon Microconcentrator with a 30-kDa molecular weight cutoff, and analyzed by EMSA using the 38-bp photoaffinity probe (lane 2). Unfractionated nuclear extract was included for comparison (lane 1). C, polypeptide composition of the immunopurified complex. The immunopurified complex (lane 2) and lower complex from unfractionated nuclear extract (lane 1) were UV-cross-linked to the photoaffinity probe and analyzed by SDS-PAGE as described under ``Materials and Methods.''



Based on the results of the immunodepletion experiment above, the lower complex from nuclear extracts of TNF-alpha-treated HUVECs was immunopurified using agarose-conjugated p50 antibody as an affinity matrix, and the bound material eluted with p50 peptide. When analyzed by EMSA, the eluted material yielded a single major retarded complex whose mobility was similar to that of the lower complex (Fig. 9B). When cross-linked to the photoaffinity probe and subjected to SDS-PAGE, the immunopurified complex was found to contain similar cross-linked polypeptides as the ``native'' lower complex from unfractionated nuclear extracts (Fig. 9C). These results clearly established that p50 was the smaller cross-linked polypeptide of the lower complex. Therefore, we concluded that the lower complex was a p65/p50 heterodimer bound to the NF-kappaB site.

Overexpression of p65 and p50

CMV-driven expression vectors for p65 (pRc/CMVp65) and p50 (pRc/CMVp50) (50 or 250 ng) were individually transfected into HUVECs along with an ICAM-1 reporter construct. The effect of overexpressing p65 or p50 was monitored using two different sets of reporter constructs: 1) the full-length constructs pGL 1.3 and pGL 1.3kappaB (Fig. 10A) and 2) the minimal promoter constructs pGL2 Prom 38bpwt and pGL2 Prom 38bpkappaB (Fig. 10B). With both sets of reporter constructs, p65 overexpression resulted in transactivation in a NF-kappaB site-dependent manner. In contrast, p50 overexpression did not transactivate either of the two wild-type reporter constructs regardless of the amount of p50 expression vector transfected into the cells and was indistinguishable from the empty expression vector, pRc/CMV (data not shown).


Figure 10: Functional effects of p65 and p50 overexpression. HUVECs were transfected with the indicated amounts of p65 (pRc/CMV p65) or p50 (pRc/CMV p50) expression vectors, 0.5 µg of luciferase reporter construct, 1 µg of pRSV-lacZ, and pBR322 to a total of 2 µg of DNA/well. Cells were lysed 16 h after transfection and assayed for luciferase and beta-galactosidase activities. Luciferase activity was normalized to beta-galactosidase activity. Induction values were expressed as the ratio of normalized luciferase activity in cells transfected with and without the indicated expression vector. Experiments shown were representative of three independent transfections carried out in triplicate. A, reporter constructs: pGL 1.3 (filled bars) and pGL 1.3kappaB (open bars). B, pGL2 Prom 38bpwt (filled bars) and pGL2 Prom 38bpkappaB (open bars), described under ``Materials and Methods.''



To determine the effect of overexpressing both p65 and p50 in the same cell, we co-transfected p65 and p50 expression vectors into HUVECs along with the reporter pGL2 Prom 38bpwt. The amount of one expression vector was varied while the other was held constant. In the first set of transfections, the concentration of the p50 expression vector was held constant and increasing amounts of pRc/CMV p65 or the empty expression vector, pRc/CMV, were transfected into HUVECs (Fig. 11A). Increasing amounts of the p65 expression vector transactivated the reporter construct in a concentration-dependent manner, whereas the control expression vector had no effect. In the converse experiment, the amount of the p65 expression vector was held constant and increasing amounts of control or p50 expression vector were transfected into the cells. In addition, an expression vector for IkappaBalpha (pRc/CMV Mad3) was included as a control (Fig. 11B). The control vector had no effect on p65-mediated transactivation, whereas increasing amounts of p50 or IkappaBalpha expression vector strongly suppressed transactivation. Using the full-length construct, pGL 1.3, similar results were obtained except that p50 could only partially suppress p65 activation at the concentrations tested (data not shown). Thus, under no condition was p50 found to augment transcription induced by p65.


Figure 11: Functional effects of p65 and p50 coexpression. HUVECs were transfected with 0.5 µg of pGL2 Prom 38bpwt, 1 µg of pRSV-lacZ, 50 ng of one expression vector (pRc/CMV p65 or pRc/CMV p50), and increasing amounts of the other (0, 50, 100, 200, and 400 ng), and pBR322 to bring the total amount of DNA to 2 µg. Luciferase activity was determined and normalized to beta-galactosidase activity. Values are given in normalized relative light units (RLU). A, cells were transfected with 50 ng of pRc/CMV p50 alone, or with increasing amounts of pRc/CMV p65, or the empty expression vector, pRc/CMV, as indicated. B, cells were transfected with 50 ng of pRc/CMV 65 alone or with increasing amounts of pRc/CMV, pRc/CMV p50, or the IkappaBalpha expression vector, pRc/CMV MAD3, as indicated.




DISCUSSION

We began our studies on ICAM-1 transcriptional regulation by determining the transcription start site of the ICAM-1 gene in HUVECs. Primer extension analysis of total RNA isolated from TNF-alpha and PMA treated cells clearly demonstrated that transcription began 40 and 41 bp upstream from the start of translation. These two start sites were 25 and 24 base pairs downstream, respectively, of the proximal TATA box identified earlier by others(15, 16) . We were unable to locate the start site of basal transcription in unstimulated cells, probably because basal ICAM-1 message levels were below the limit of detection (14) . However, no alternative start sites were detected, even under conditions known to stabilize ICAM-1 mRNA in HUVECs(14) , such as treatment with PMA or cycloheximide, (^2)suggesting that basal and induced transcripts may be initiated from the same start site. Other studies on the transcriptional start site(s) of the ICAM-1 gene have been published, although they were conducted in other cell types(15, 16, 17) . In human keratinocytes and A431 cells, a human epidermoid squamous cell carcinoma line, stimulated with IFN-, ICAM-1 transcription was found to begin at -39 and -40 relative to the start of translation(15) . Voraberger et al.(16) found two start sites in Hs913T fibrosarcoma cells at 319 and 41 bp upstream from the translational start site. They also reported differential use of these sites in A549 cells in response to TNF-alpha and PMA. ICAM-1 transcripts in unstimulated and TNF-alpha stimulated A549 cells were initiated at -41, whereas PMA-induced transcription began at -319. Another start site was detected at -84 in the Burkitt lymphoma cell line Raji(17) . Therefore, basal and induced ICAM-1 transcription have been reported to initiate at multiple sites in different cell types, but we found no evidence for this in endothelial cells.

In this study, we identified a single NF-kappaB site within a 92-bp responsive region that was essential for the ICAM-1 promoter to respond to TNF-alpha, IL-1beta, PMA, and LPS in endothelial cells. van de Stolpe et al.(20) recently identified this site based on deletion analysis of the ICAM-1 5`-flanking sequence in human 293 embryonal kidney cells. Their evidence suggested that this NF-kappaB site was largely responsible for mediating PMA and TNF-alpha responsiveness of the ICAM-1 promoter in this cell type. The ICAM-1 NF-kappaB site, TGGAAATTCC, deviates from the published NF-kappaB consensus sequence of GGGRNNYYCC at the extreme 5` end of the sequence where the conserved guanine residue is replaced by a thymine residue(46) . Although this type of substitution within the NF-kappaB consensus sequence is uncommon, it has been observed previously in the promoters of the urokinase, G-CSF, GM-CSF, tissue factor, and IL-8 genes(53, 54, 55, 56, 57) . It has been suggested that NF-kappaB sites differing at this base can selectively recruit other members of the NF-kappaB/rel family besides the classical p65/p50 heterodimer(58) .

Apart from an A to T transition, the IL-8 NF-kappaB site, TGGAATTTCC, is identical to the ICAM-1 NF-kappaB site, and our studies on the ICAM-1 kappaB site parallel those obtained with the IL-8 promoter kappaB site. Both elements bound p65 homodimers and p65/p50 heterodimers when nuclear extracts from TNF-alpha-treated cells were analyzed by EMSA(59, 60) . In co-transfection experiments, p65 and p50 did not synergistically transactivate reporter constructs containing the IL-8 or ICAM-1 kappaB sites(59) . Rather, in both cases, increasing amounts of p50 expression vector resulted in a substantial decrease in p65-mediated transactivation. In contrast, co-expression of p50 and p65 synergistically transactivated reporter constructs containing the human immunodeficiency virus or IL-2 receptor kappaB sites, which do not deviate from the NFkappaB consensus sequence(61, 62) . These results suggest that activation of the IL-8 and ICAM-1 promoters through their respective kappaB sites depends primarily on p65 homodimers. This is further supported by the finding that antisense oligonucleotides to p65, but not p50, completely inhibited PMA-stimulated IL-8 production (59) .

The IL-8 kappaB site was found to cooperatively interact with an adjacent C/EBP site, resulting in the synergistic activation of the IL-8 promoter(51, 60) . Considering the high degree of similarity between the IL-8 and ICAM-1 kappaB sites, we anticipated that the potential C/EBP site adjacent to the ICAM-1 kappaB site might also be important. EMSAs using C/EBP specific antibodies and unlabeled competitors containing the ICAM-1 C/EBP site did not suggest the involvement of C/EBP in inducible complex formation. However, since C/EBP involvement in transcriptional regulation may not be readily apparent in gel shift experiments, we cannot rule out the possibility of a C/EBP/NF-kappaB interaction until functional studies using a mutated C/EBP site are performed.

The promoters for two other cytokine-inducible adhesion molecules, VCAM-1 and E-selectin, have been examined in detail(21, 22, 23, 24, 25, 26, 63) . Unlike the single essential NF-kappaB site present in the ICAM-1 promoter, the VCAM-1 and E-selectin promoters contained two and three critical kappaB sites, respectively. Mutation or deletion of these essential kappaB elements rendered their respective promoters unresponsive to cytokine stimulation in endothelial cells. The five kappaB sites from these two promoters have different decameric sequences, but all five possess the characteristic triplet of guanine residues present at the 5` end of the NF-kappaB consensus sequence. In contrast to ICAM-1 reporter constructs, co-expression of p65 and p50 augmented rather than inhibited transactivation of the E-selectin and VCAM-1 reporter constructs(48, 49, 64) , providing functional evidence that p65/p50 heterodimers may be involved in the regulation of these two promoters. Therefore, all three adhesion molecule promoters critically rely on the presence of NF-kappaB sites, but the ICAM-1 gene differs from the E-selectin and VCAM-1 genes, in that it appears to be selectively activated by p65 homodimers.

Considering the pathophysiological importance of ICAM-1, E-selectin, and VCAM-1 in the progression of inflammatory responses, these adhesion proteins have become attractive therapeutic targets(3) . Despite an enormous effort to identify small molecule inhibitors of cell adhesion, little progress has been made in this area. One relatively untapped aspect of cellular adhesion is the transcriptional regulation of these inducible genes. Dissection of the ICAM-1, E-selectin, and VCAM-1 promoters has identified specific elements that confer responsiveness to inflammatory cytokines in endothelial cells. Although cytokine up-regulation of each promoter is distinct, all three promoters depend on the presence of upstream NF-kappaB sites, suggesting that selective inhibitors of NF-kappaB might be particularly useful in the treatment of acute and chronic inflammatory diseases.

In summary, our findings indicate that TNF-alpha-induced transcription of the ICAM-1 gene in endothelial cells is directed exclusively from the proximal TATA box and critically depends on a single atypical NF-kappaB located 187 bp upstream of the transcription start site. Transcriptional activation by IL-1beta and LPS, and to a lesser extent PMA, is also mediated through this element. Even though endogenous p65 homodimers and p65/p50 heterodimers bind to the ICAM-1 kappaB site, functional studies implicate the p65 homodimer as the key transactivator working through this site on the ICAM-1 promoter.


FOOTNOTES

*
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.

§
Present address: GeneMedicine, Inc., 8080 North Stadium Dr., Suite 2100, Houston, TX 77054-1823.

To whom correspondence should be addressed: Dept. of Inflammatory Diseases, Boehringer Ingelheim Pharmaceuticals, Inc., 175 Briar Ridge Rd., Ridgefield, CT 06877. Tel.: 203-798-5127; Fax: 203-791-6468.

(^1)
The abbreviations used are: ICAM-1, intercellular adhesion molecule-1 (CD54); LFA-1, lymphocyte function-associated antigen-1 (CD11a/CD18); Mac-1, CD11b/CD18; LPS, lipopolysaccahride; TNF-alpha, tumor necrosis factor-alpha; IL-1beta, interleukin-1beta; IFN-, interferon-; mRNA, messenger RNA; HUVEC, human umbilical vein endothelial cell; CAT, chloramphenicol acetyltransferase; PMA, phorbol 12-myristate 13-acetate; E-selectin, CD62E; VCAM-1, vascular cell adhesion molecule-1 (CD106); NF-kappaB, nuclear factor kappaB; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; DTT, dithiothreitol; BrdUrd, 5-bromo-2`-deoxyuridine 5`-triphosphate; PAGE, polyacrylamide gel electrophoresis; C/EBP, CAAT/enhancer-binding protein; bZIP, basic coiled-coil; ATF, activating transcription factor; CREB, cyclic AMP response element binding protein; G-CSF, granulocyte colony stimulating factor; GM-CSF, granulocyte/macrophage colony-stimulating factor; IL-8, interleukin-8; bp, base pair(s).

(^2)
H. C. Ledebur and T. P. Parks, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. C. Stratowa of the Ernst Boehringer Institute, Bender & Co., Vienna, Austria, for the plasmids pBHluc1.3 and p5`Delta981; Dr. Patrick Baeuerle of the Biochemistry Institute, Albert-Ludwigs University, Freiburg, Germany, for the expression vectors pRc/CMV p65, pRc/CMV p50, and pRc/CMV Mad3; and Dr. John Miglietta for the plasmid pGEMJJM used in this study. We also thank Dr. C. Myers and H. Smith for assistance with cell culture, Dr. J. Miglietta, G. Hansen, and A. Shrutkowski for DNA sequence analysis, and L. Rondano for preparing the figures.


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