Inhibition of Nuclear Factor-B–Mediated Adhesion Molecule Expression in Human Endothelial Cells

From the Cardiology Section (J.M.L., J.S.C., W.L.A.-L., A.J.B.) and Nephrology Section (T.H.), Department of Medicine, and the Molecular and Cellular Biology Graduate Program (J.M.L., J.S.C.), Tulane University Medical School, and the VA Medical Center (T.H.), New Orleans, La.

Correspondence to Jean M. Lockyer, PhD, Cardiology Section SL48, Tulane University Medical School, 1430 Tulane Ave, New Orleans, LA 70112-2699. E-mail Lockyer.Jean{at}tmc.tulane.edu

	Abstract

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Abstract—The transcriptional regulatory protein nuclear factor-B (NF-B) participates in the control of gene expression of many modulators of the inflammatory and immune responses, including the adhesion molecules E-selectin and intercellular adhesion molecule-1 (ICAM-1). NF-B is found in the cytoplasm complexed with its inhibitory protein IB. On activation, IB is phosphorylated and degraded, thereby freeing NF-B for translocation to the nucleus. We have generated populations of endothelial cells expressing wild-type and a proteolysis-resistant mutation of IB that is lacking the 36 N-terminal amino acids (IBN) in order to examine the effects of expression of the mutated IB on tumor necrosis factor- (TNF-)–induced E-selectin and ICAM-1 expression. Wild-type and IBN were introduced into primary endothelial cells using retrovirus infection followed by selection with G418. The IBN protein remained at untreated control levels in endothelial cells treated with TNF- and also remained complexed with the NF-B family member p65. Furthermore, TNF-–induced NF-B DNA binding activity was inhibited in the population of endothelial cells expressing IBN. That population of cells was also refractory to upregulation of E-selectin and ICAM-1 after treatment with TNF-. The use of a truncated IB protein to prevent NF-B–mediated gene expression provides a novel and specific approach for investigating the role of NF-B in processes associated with adhesion molecule expression during inflammation.

Key Words: nuclear factor-B • vascular endothelium • adhesion molecule • inflammation • gene expression

	Introduction

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The endothelial cell inflammatory response is believed to play a central role in the pathogenesis of atherosclerosis and thrombosis and is also considered to be a pivotal factor in myocardial reperfusion injury by mediating the migration of neutrophils into ischemic/reperfused myocardium. We are presently examining the role of the transcriptional activator NF-B in upregulating the expression of the endothelial cell adhesion molecules ICAM-1 and E-selectin, which are believed to play pivotal roles in neutrophil and monocyte adhesion to activated endothelium (reviewed in Reference 1¹ ).

A number of reports have examined the role of NF-B in the activation of adhesion molecule expression in cultures of HUVECs. Analyses of the promoter regions of E-selectin,^{2 3 4} VCAM-1,^{5 6 7} and ICAM-1⁸ have indicated that NF-B plays a pivotal role in cytokine-induced expression. Further studies using reagents such as proteasome and protease inhibitors,^{2 6} which prevent progression of the NF-B activation pathway, have also prevented cytokine-induced activation of the endogenous E-selectin, VCAM-1, and ICAM-1 genes. NF-B activation and subsequent adhesion molecule expression have also been prevented in endothelial cells by using an adenovirus vector to overexpress wild-type IB protein.⁹ The focus of the present study was to develop a retrovirally transduced stable endothelial cell model to address the hypothesis that overexpression of an NF-B dominant-negative inhibitory IB protein that is resistant to cytokine-induced proteolysis will constitutively inhibit NF-B activation in response to cytokine treatment and thereby decrease NF-B–dependent gene activity and TNF-–induced endothelial adhesion molecule expression.

NF-B is present in most cells in a cytosolic form that is complexed with an inhibitor, IB,^{10 11 12} that prevents NF-B from being translocated to the nucleus and binding to DNA. Activation of NF-B involves release of the protein from IB after signals that stimulate proteolysis of IB (reviewed in References 13 and 14^{13 14} ). Many inducers of NF-B function through a major activation pathway that results in phosphorylation at serine residues 32 and 36 contained within the N-terminal region of the protein.^{15 16 17 18 19} Phosphorylation leads to ubiquitination and subsequent degradation by the 26S proteasome unit (Reference 14¹⁴ and references therein). Recently, there have been reports of dominant-negative mutations of IB that are not phosphorylated and are therefore not proteolyzed.^{15 17 20} One such protein is a mutant IB that is lacking the N-terminal 36 amino acids. The serine residues are not present, and the IB protein remains complexed with NF-B and prevents translocation to the nucleus.¹⁷

A mutated IB protein that was deleted at the N-terminus was used to examine endothelial adhesion molecule expression in primary cultures of HUVECs. Retroviruses containing wild-type IB (IBwt) or a mutated IB that is truncated at the N-terminal 36 amino acids (IBN) were used to infect primary cultures of HUVECs. After infection, the cells that integrated the retrovirus were selected with G418. Analysis of the retroviral IB proteins showed that the truncated IB was not degraded after treatment of the cells with TNF- and that NF-B activation was inhibited. Furthermore, the population of endothelial cells that overexpressed the mutant IB was refractory to cytokine-induced upregulation of E-selectin and ICAM-1 gene expression. This novel population of endothelial cells represents a unique tool for the evaluation of NF-B function in response to acute inflammatory stimuli in endothelial cells.

	Materials and Methods

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Cells
Cell culture reagents were purchased from GIBCO-BRL unless otherwise noted. HUVECs were isolated using type II collagenase (Worthington). Briefly, umbilical cords were obtained, and the veins were washed with PBS, followed by an infusion of 0.1% collagenase for 20 minutes at 37°C. The resulting cell suspension was removed, and the pelleted cells were plated onto tissue culture flasks coated with 0.2% gelatin. The cells were maintained in complete medium 199, ie, medium 199 supplemented with 10% FBS, 60 µg/mL endothelial cell growth supplement (Collaborative Research), 100 µg/mL heparin, and antibiotics (100 µL/mL penicillin and 100 µg/mL streptomycin). The cells were passaged at confluence at a ratio of 1:3 or 1:4. The isolations were examined for endothelial morphology and assessed for the expression of FVIII-RA by immunofluorescence to confirm endothelial identity.

Immunofluorescence was performed using a specific rabbit polyclonal antibody (Dako). Endothelial cells were plated at a density of 30 000 per well onto gelatin-coated six-well tissue culture plates. The following day, the confluent monolayers were incubated for 45 minutes at 37°C with a 1:100 dilution of FVIII-RA antibody in complete medium 199. Control cultures were incubated with an identical amount of normal rabbit serum. The cells were then rinsed two times with PBS and incubated for 45 minutes at 37°C with a 1:200 dilution of goat anti-rabbit FITC-conjugated antibody (Sigma Chemical Co). The cells were then washed once with complete medium 199 and two times with PBS; after which, 1 mL of 30 µmol/L EDTA in PBS without calcium and magnesium was added to each well and incubated for 10 minutes at 4°C. The cells were collected from each well and transferred to cuvettes, and the amount of fluorescence was determined on a Fluoromax-2 fluorescent spectrophotometer (Instruments S.A. Inc). The emission slits were set at 490-nm excitation and 520-nm emission. Each cell population was analyzed in triplicate. Human coronary artery smooth muscle cells (Clonetics) were used as negative control cells.

Plasmid Construction
The ß-gal and IB retroviral constructs were made using the pLNCX vector.²¹ The pLNCXß-gal retroviral plasmid was constructed by digesting pCMVß-gal²² with NotI to obtain the ß-gal gene. The fragment was then treated with Klenow to generate blunt ends and subcloned into the HpaI site of pLNCX. Orientation was determined by restriction mapping with ClaI. The IBwt and IBN fragments were obtained by digesting pCMV4IBwt¹⁷ and pCMV4IBN¹⁷ (gift of Dean Ballard, Vanderbilt University, Nashville, Tenn) with HindIII and SmaI. The inserts were then subcloned into pLNCX digested with HindIII and HpaI.

Preparation of Retroviruses
The viruses were generated using a two-step packaging protocol. The first packaging cell line, 2, was plated at a density of 150 000 per 60-mm plate. Eighteen hours later, the cells were transfected with pLNCX plasmids (10 µg of DNA plus 10 µL of lipofectin reagent) containing the control ß-gal or the IB-encoding cDNAs (IBwt and IBN). Five hours after treatment, the medium was replaced with growth medium (DMEM plus 10% fetal calf serum). Thirty-six hours after lipofection, the medium was harvested and used to infect the second packaging cell line, PA317 cells (obtained from American Type Culture Collection). The PA317 cells were plated at a density of 150 000 per 60-mm plate 16 to 18 hours before exposure to the 2 supernatants (100 µL to 1 mL). Incubation was performed in the presence of 4 µg/mL polybrene. Twenty-four hours after exposure to the 2 supernatants, the PA317 cells were replated onto 100-mm plates and selected with G418 at a concentration of 750 µg/mL active G418. Colonies of virus-producing cells were then pooled and used as a source of virus for transduction of HUVECs. The viruses were titered as described²¹ using NIH3T3 cells. All viruses used for these experiments had titers of at least 10⁵/mL.

Transduction of Endothelial Cells
Newly isolated cells from umbilical cords were plated onto gelatin-coated T-25 flasks. On reaching 75% to 100% confluence (within 5 days of isolation), the cells were plated at a density of 100 000 to 200 000 per 60-mm plate and used for transduction with IB or control retroviruses. Viral transductions were performed as follows. Twenty-four hours after plating, the cells were exposed for 1 minute to 250 µg/mL DEAE-dextran in PBS. The DEAE-dextran was then aspirated and replaced with 1 to 2 mL of viral supernatant in a total volume of 3 mL of heparin-free endothelial cell growth medium 199. After 4 hours, the virus-containing medium was removed, and the cells were incubated in fully supplemented endothelial cell growth medium 199. Twenty-four hours subsequent to the first exposure to viral supernatant, the cells were incubated with a second aliquot of viral supernatant as described above. Twenty-four hours after the second exposure, the cells were replated onto 100-mm gelatin-coated plates and selected with G418 at a concentration of 400 µg/mL active G418. After expansion of the G418-selected populations, the cells were maintained in 200 µg/mL active G418. The G418-selected endothelial cells were not used beyond passage 6. Characterization of FVIII-RA expression was conducted at passage 3 or 4.

Western Blot Analysis
Chemical reagents for the protein analyses were purchased from Sigma. The acrylamide and cross-linking reagents for the polyacrylamide gels were purchased from Bio-Rad. Whole-cell extracts for Western blot analysis were prepared by lysing cells in 2x protein sample buffer (10% glycerol, 2.5% SDS, and 15.7 mmol/L Tris, pH 6.8). Protein concentrations were obtained using the Pierce BCA assay. Electrophoresis was performed on 12% SDS–polyacrylamide gels; after electrophoresis, the proteins were transferred to nitrocellulose. IB was detected using the IB antibody SC-847 from Santa Cruz Biotechnology. The endogenous IB protein is distinguishable from the retrovirally encoded IBwt or IBN proteins by its position of migration in the polyacrylamide gels. Generation of the FLAG epitope–containing full-length IB results in a protein that migrates at a slightly higher molecular weight compared with the endogenous endothelial IB. The IBN protein, which also possesses the FLAG epitope at the N-terminus, migrates as a band smaller than the endogenous IB because of the deletion of the 36 amino acids from the N-terminus of IB.

Immunoprecipitations
Immunoprecipitations used the FLAG epitope²³ incorporated into the N-terminus of the wild-type and N IB proteins.¹⁷ Cytosolic extracts were prepared as described.²⁴ Briefly, cells were washed in Tris-buffered saline and pelleted at 4°C. The pellet was resuspended in cold buffer A (10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L DTT, and 0.5 mmol/L PMSF) supplemented with a cocktail of protease inhibitors (antipain, aprotinin, leupeptin, pepstatin A, and soybean trypsin inhibitor) at a final concentration of 5 µg/mL. After a 15-minute incubation on ice, the detergent Nonidet NP-40 was added to a concentration of 0.1%, and the cells were vortexed vigorously and pelleted. The supernatant was then equilibrated to 50 mmol/L HEPES, 250 mmol/L NaCl, and 5 mmol/L EDTA (final concentration). The FLAG epitope–containing protein was then immunoprecipitated by incubation with the anti-FLAG monoclonal antibody M2 (IBI-Kodak) conjugated to agarose beads. The immunoprecipitation was performed by incubating the cell lysates with the beads at 4°C for 1 hour with gently mixing. The beads were then pelleted and washed three times in equilibration buffer. After the final wash and a gentle spin to remove the beads, the supernatant was removed, and the beads were resuspended in SDS sample loading buffer and heated for 5 minutes at 100°C. The supernatant was then used for Western blot analysis on a 12% denaturing SDS-polyacrylamide gel. The retrovirally encoded IB proteins were detected using the SC-847 antibody. Coprecipitated NF-B family members were detected with a specific rabbit polyclonal antibody that recognizes NF-B p65 (Santa Cruz Biotechnology). Western blots were developed using an alkaline phosphatase–conjugated secondary antibody (Bio-Rad).

Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared from endothelial cell monolayers using a high-salt extraction procedure with modifications.²⁵ Cells were harvested by scraping and washed in 0.5 vol cold PBS. The cells were then washed once in 0.1 vol cold buffer A (10 mmol/L HEPES [pH 7.9], 1.5 mmol/L MgCl₂, 10 mmol/L KCl, and 0.5 mmol/L DTT). The washed cell pellets (corresponding to the cells derived from one confluent T-75 flask) were then resuspended in 50 µL of buffer A plus 0.1% Nonidet NP-40 supplemented with 1 µg/mL of leupeptin and aprotinin and incubated on ice for 10 minutes. After incubation, the pellets were mixed briefly by vortexing and centrifuged at 10 000 rpm at 4°C for 5 minutes in a microcentrifuge. The supernatant was carefully removed, and the nuclear pellet was resuspended in 20 µL of cold buffer C (20 mmol/L HEPES, 25% glycerol, 0.42 mol/L NaCl, 1.5 mmol/L MgCl₂, 0.2 mmol/L EDTA, 0.5 mmol/L DTT, and 0.5 mmol/L PMSF) containing 1 µg/mL of leupeptin and aprotinin and incubated on ice for 15 minutes with intermittent vortexing. The extracts were then centrifuged at 10 000 rpm at 4°C for 10 minutes, and the supernatant was aliquoted and frozen at -70°C. Protein concentrations were determined using the Bio-Rad protein assay kit.

Gel-shift analyses were performed as described using probes containing the B binding site from the mouse light chain enhancer or an NF-B site from the ICAM-1 promoter. The duplex B probe²⁶ was generated by annealing the two strands (5'AGCTTCAGA GGGGAC TTTCCGAGAGG 3' and 5' TCGA CCTCTCGGAAA GTCCCCTCTGA 3') and then radiolabeling with [-³²P]dCTP (Dupont) using the Klenow fragment of DNA polymerase I. The ICAM-1 NF-B probe was generated by annealing two complementary oligonucleotides corresponding to positions -177 to -189 of the ICAM-1 promoter⁸ and end-labeling with [-³²P]ATP using T4 kinase. Binding reactions (20 µL) containing 10 µg of nuclear extract, 2 µg poly(dI-dC) in 10 mmol/L Tris (pH 7.5), 40 mmol/L NaCl, 4 mmol/L DTT, 1 mmol/L EDTA, and 4% glycerol were incubated for 30 minutes at 4°C. Binding was initiated by the addition of 10⁴ cpm of ³² P-labeled oligonucleotide probe (0.1 to 0.2 ng of DNA) for 15 minutes at room temperature. Products were resolved on a 4% polyacrylamide gel in 1x TBE (45 mmol/L Tris-borate-EDTA) buffer.

Immunofluorescence Analysis
Cells were plated in triplicate at a density of 30 000 per well onto gelatin-coated six-well tissue culture plates. The following day, the confluent cultures were treated with 10 ng/mL of TNF- for 4 hours at 37°C or used as untreated controls. After treatment, the cells were analyzed by immunofluorescence for surface expression of ICAM-1 and E-selectin. Fluorescence measurements were obtained as described above for the analysis of FVIII-RA cell surface expression. Briefly, the control or treated cells were incubated with the mouse monoclonal ICAM-1 primary antibody RR1 (BIOSOURCE) at a concentration of 0.5 µg/mL or the E-selectin mouse monoclonal antibody (PharMingen) at 1 µg/mL. Negative controls were incubated with an IgG1 isotype control antibody. The FITC-conjugated goat anti-mouse secondary antibody (Sigma) was used at a 1:160 dilution.

	Results

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In order to examine the effects of overexpression of IB on cytokine-induced activation of ICAM-1 and E-selectin gene expression, populations of retrovirally transduced endothelial cells that expressed a control ß-gal, IBwt, or IBN were produced. The retroviral constructs were made by subcloning control ß-gal cDNA or FLAG-tagged IB cDNAs¹⁷ into the pLNCX vector and then generating the retroviruses using the PA317 packaging cell line.

Endothelial cells were then transduced with ß-gal, IBwt, or IBN retroviruses, followed by selection with G418 and expansion of the resistant population. The transduction efficiencies were estimated to be no more than 10% to 50% but allowed for adequate expansion of the population within a month of infection of the endothelial cells.

Expression and Stability of IB in Virally Transduced Primary HUVECs
In order to examine the expression levels and the stability of the retrovirally encoded IB proteins, Western blot analyses were performed after treatment with TNF-. The G418-resistant HUVECs that had been transduced with IBwt retrovirus, IBN, or control ß-gal retrovirus were treated with TNF- for 30 minutes. Protein extracts (30 µg) were then probed with an antibody to IB. The results shown in Fig 1 indicate that the overall expression levels of the IBwt and IBN proteins were similar in the two cell populations (lanes 3 and 5). The retrovirally encoded IB proteins also appeared to be expressed at higher levels relative to the endogenous IB protein indicated by the asterisk in lane 1 and also present in lanes 3 and 5. Furthermore, on treatment with TNF-, the endogenous IB was rapidly degraded as was the retrovirally expressed IBwt (lane 3 versus 4). The IBN, however, was not degraded (lanes 5 and 6). IBwt protein, which was continuously expressed under these conditions, approached untreated levels after 2 hours of TNF treatment (data not shown).

View larger version (44K): [in this window] [in a new window]   Figure 1. Western blot of IB protein expression in endothelial cells. Thirty micrograms of protein extract prepared from G418-selected HUVECs transduced with control ß-gal (lanes 1 and 2), IBwt (lanes 3 and 4), or IBN (lanes 5 and 6) retroviruses was analyzed on Western blots. - indicates untreated cells; +, 30 minutes of treatment with 10 ng/mL TNF-. The asterisk indicates the endogenous IB protein (lane 1), which is also observed in lanes 3 and 5.

To further evaluate the stability of the expressed IB proteins, samples were also prepared from cells that were exposed to cycloheximide in order to prevent protein synthesis (Fig 2). Before treatment with TNF-, the cells were treated with 50 µg/mL of cycloheximide for 1 hour and then incubated with TNF- for time periods of up to 2 hours. As anticipated, IBN remained at levels equivalent to those observed in untreated cells for the 2-hour period examined (lanes 5 to 8). The retrovirally encoded IBwt (lanes 1 to 4), however, was rapidly degraded as was the endogenous IB (indicated by the asterisk in lane 1 and also present in lane 5.) This experiment demonstrated that the IBN protein was stable in TNF-–treated human endothelial cells.

View larger version (38K): [in this window] [in a new window]   Figure 2. Expression of IB in TNF-–treated HUVECs pretreated with cycloheximide. Protein extracts prepared from TNF-–treated HUVECs that had been pretreated with 50 µg/mL cycloheximide for 1 hour were analyzed on Western blots. The endothelial cell populations analyzed expressed IBwt (lanes 1 to 4) or IBN (lanes 5 to 8). The time points indicate the length of exposure to 10 ng/mL TNF-. The asterisk (lane 1) designates the endogenous IB protein, which is also observed in lane 5.

Coimmunoprecipitation
In order to demonstrate that IBN retained the ability to complex with NF-B family members on treatment of endothelial cells with TNF-, a coimmunoprecipitation experiment was performed using the FLAG epitope present on the IB proteins to isolate IBwt, IBN, and other proteins to which they were complexed. Confluent monolayers of the virally transduced and selected HUVECs (control ß-gal, IBwt, or IBN) were treated for 45 minutes with 5 ng/mL TNF-. After treatment, cytosolic extracts were prepared, and the FLAG epitope–containing protein was isolated by incubation with the anti-FLAG monoclonal antibody M2 conjugated to agarose beads. The immunoprecipitated and coprecipitated proteins were then analyzed on Western blots.

Fig 3 shows the results of Western blots using the IB antibody (Fig 3A) and an antibody to the NF-B family member p65 (Fig 3B) to identify the isolated proteins. IBwt protein was undetectable 45 minutes after TNF- treatment (Fig 3A, lane 4) whereas IBN protein was still present (Fig 3A, lanes 5 and 6). In Fig 3B, IBN (lanes 5 and 6) remained associated with NF-B p65 45 minutes after treatment with TNF-. The p65 protein was also detectable in immunoprecipitates derived from IBwt-expressing cells 45 minutes after treatment (Fig 3B, lane 4), although the amount was greatly reduced compared with that in the untreated control cells (lane 3).

View larger version (50K): [in this window] [in a new window]   Figure 3. Western blot of immunoprecipitated and coprecipitated products isolated from transduced endothelial cells using an anti-FLAG epitope antibody. Panel A shows the products isolated from G418-selected HUVECs transduced with control ß-gal (lanes 1 and 2), IBwt (lanes 3 and 4), or IBN (lanes 5 and 6) retroviruses and probed with an IB antibody. - indicates untreated cells; +, 45 minutes of treatment with 10 ng/mL TNF-. Panel B depicts immunoprecipitated products from the same immunoprecipitation shown in panel A but probed with an NF-B p65-specific antibody.

Characterization of Endothelial Cell Phenotype in Retrovirally Transduced Cell Populations After G418 Selection
In order to demonstrate that the G418-selected endothelial cells retained phenotypic features of HUVECs, cells transduced with the IBwt virus (HUVEC-wt) or IBN retrovirus (HUVEC-N) that had undergone selection and expansion were analyzed for endothelial morphological phenotype and for the expression of the FVIII-RA surface protein. The endothelial cells expressing IBwt and IBN (Fig 4B and 4C) were visually indistinguishable from normal HUVECs (Fig 4A) as were those cells transduced with the ß-gal virus (data not shown). The endothelial cell populations also retained surface expression of FVIII-RA at levels comparable to control HUVECs that had not been transduced with retrovirus (Fig 5). Human coronary artery smooth muscle cells were also included in the analysis as a negative control.

View larger version (87K): [in this window] [in a new window]   Figure 4. Morphological assessment of retrovirally transduced HUVECs after G418 selection and expansion of the populations. Endothelial cells were plated onto gelatin-coated 60-mm tissue culture plates. Photomicrographs of confluent cultures of normal HUVECs (A), HUVECs transduced with IBwt retrovirus (B), or HUVECs transduced with the IBN retrovirus (C) are shown. Magnification is 40-fold. The cells were plated at passage 4 (control HUVECs) or passage 5.

View larger version (25K): [in this window] [in a new window]   Figure 5. Expression of FVIII-RA in transduced HUVECs after selection with G418 and expansion of the populations. The relative levels of surface FVIII-RA were assessed using immunofluorescence analysis. Confluent cultures of HUVECs or HUVECs transduced with the IB retroviruses (HUVEC-IBwt and HUVEC-IBN) were incubated with a specific rabbit polyclonal antibody to FVIII-RA or normal rabbit serum (NRS) control. Fluorescence was determined using an FITC-conjugated goat anti-rabbit antibody. Human coronary artery smooth muscle cells (HCASMs) were used as negative control cells. Values for FVIII-RA levels were determined in quadruplicate. HUVEC-IBwt and HUVEC-IBN were plated at passage 5, and the control HUVECs (obtained from a different isolation) were plated at passage 3.

Inhibition NF-B DNA Binding Activity
In order to demonstrate that expression of the deletion mutant effectively inhibited NF-B DNA binding activity in TNF-–treated HUVECs, electrophoretic mobility shift assays were performed on crude nuclear extracts derived from stable transfectants expressing control ß-gal gene, IBwt, or IBN. The results in Fig 6 show that NF-B DNA binding activity was present in nuclear extracts from the TNF-–treated control ß-gal cells (Fig 6A and 6B, lane 2). TNF-–induced NF-B DNA binding activity was also observed in nuclear extracts from IBwt-expressing cells (Fig 6A and 6B, lane 2), although at reduced levels compared with the control population of cells. Those cells that expressed IBN, however, showed no evidence of activation of NF-B DNA binding after treatment with TNF- using either the B light chain enhancer probe or the ICAM-1 NF-B probe. A similar result was also obtained using a duplex containing the NF-B site from the E-selectin promoter (data not shown). These results indicate that expression of the IBN protein in an endothelial cell population potently inhibits TNF-induced NF-B DNA binding activity.

View larger version (67K): [in this window] [in a new window]   Figure 6. NF-B DNA binding activity in transduced endothelial cells. Nuclear extracts were prepared from HUVECs transduced with the ß-gal, IBwt, or IBN retroviruses. DNA binding activity was assessed using oligonucleotide duplexes corresponding to the light chain enhancer sequence (A) or the NF-B binding site from the ICAM-1 promoter (B). Control HUVECs (A and B, lanes labeled 1) were untreated; TNF-–treated HUVECs (A and B, lanes labeled 2) were treated 45 minutes with 10 ng/mL TNF-. Lanes labeled as 3 and 4 in panel A are the same extracts incubated in the presence of 200 ng of specific competitor DNA. Lanes labeled as 3 in panel B are the TNF-–treated sample incubated in the presence of 200 ng of specific competitor DNA.

Analysis of TNF-–Induced E-Selectin and ICAM-1 Expression in IB-Expressing HUVECs
In order to determine whether overexpression of the IBwt or IBN protein had an effect on adhesion molecule expression, we examined E-selectin and ICAM-1 levels in response to treatment with TNF-. G418-selected HUVECs that were transduced with IBwt or IBN retroviruses were treated with 10 ng/mL TNF- for 4 hours. Cells were then analyzed for the presence of E-selectin or ICAM-1 expression at the endothelial cell surface. Fluorescence analysis showed that in those HUVECs expressing the full-length IB (HUVEC-IBwt), both ICAM-1 (Fig 7A) and E-selectin (Fig 7B) were upregulated to levels similar to those observed in control normal HUVECs after 4 hours of TNF- treatment. Those cells expressing the N-terminal–deleted IB (HUVEC-IBN), however, were refractory to TNF-–stimulated ICAM-1 and E-selectin expression. Furthermore, there was an indication that the basal level of ICAM-1 surface expression (Fig 7A) was also reduced in the HUVEC-IBN population compared with control HUVECs. HUVEC-IBN untreated cells showed ICAM-1 values that were equivalent to the isotype-matched IgG1 negative control values.

View larger version (25K): [in this window] [in a new window]   Figure 7. Immunofluorescent analysis of E-selectin and ICAM-1 expression in TNF-–treated HUVECs. Confluent monolayers of normal HUVECs, HUVECs expressing IBwt (HUVEC-IBwt), or HUVECs expressing the IBN protein (HUVEC-IBN) were treated with 10 ng/mL TNF- or used as untreated control cultures. Four hours after treatment, the cells were incubated with monoclonal antibodies to ICAM-1 (A), E-selectin (B), or an isotype-matched Ig61 control antibody. Fluorescence was determined using a goat anti-mouse FITC-conjugated antibody.

These data suggest that NF-B–dependent endothelial adhesion molecule expression can be effectively prevented by using a "dominant-negative" IB protein to prevent NF-B activation. Endothelial expression of an IB protein that was deleted at the N-terminus by 36 amino acids resulted in a stable NF-B/IB complex and subsequent inhibition of NF-B activation. Utilization of endothelial cells expressing the IB mutant provides a means of specifically examining the role of NF-B in inflammation-induced endothelial adhesion molecule expression.

	Discussion

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Increasing evidence indicates that activation of the endothelium during inflammation plays a role in the pathogenesis of atherosclerosis and thrombosis and is a pivotal factor in myocardial reperfusion injury, mediating the migration of neutrophils into ischemic/reperfused myocardium. Recent studies have demonstrated that activated endothelium displays upregulated levels in a variety of molecules, including growth factors, cytokines, and cell surface adhesion molecules. The upregulated endothelial cell adhesion molecules act in concert with leukocyte adhesion molecules to coordinate the localization of leukocytes to inflamed tissue (reviewed in Reference 1¹ ). We are presently examining the role of the transcriptional activator NF-B in upregulating ICAM-1 and E-selectin expression, which are believed to play pivotal roles in neutrophil and monocyte adhesion to activated endothelium. NF-B is a multisubunit transcriptional regulatory factor that participates in the control of gene expression of many protein modulators of the inflammatory and immune response (reviewed in References 27 and 28^{27 28} ). Because of its central role in acute inflammation, this transcriptional regulatory factor may provide a key target for modulating endothelial adhesion molecule expression in response to inflammatory stimuli.

Several studies^{15 16 17 18 20} have indicated that phosphorylation of serine residues 32 and 36 is a crucial event for the TNF-–induced activation of NF-B in T cells. This activation pathway is conserved on stimulation with a variety of agents, including LPS, IL-ß, or phorbol 12-myristate 13-acetate. We have extended these findings to endothelial cells to show that an IB protein lacking the N-terminal 36 amino acids is resistant to proteolysis in TNF-–treated human endothelial cells and retains the ability to complex with NF-B. Furthermore, endothelial cells transduced with retroviruses expressing the truncated IB protein do not exhibit increased surface expression of ICAM-1 or E-selectin after 4 hours of treatment with TNF- (Fig 5). The truncated IB protein therefore appears to exhibit a dominant-negative effect by preventing NF-B–mediated upregulation of E-selectin and ICAM-1 expression.

The endothelial cells transduced with the IBwt retrovirus exhibited reduced NF-B DNA binding activity compared with the control cells (Fig 6), but the reduced binding did not result in decreased levels of ICAM-1 or E-selectin expression in response to treatment with TNF- (Fig 7). The lack of phenotypic effect on NF-B–mediated gene expression by simply overexpressing IBwt may be due, at least in part, to heterogeneity of the endothelial cell populations obtained by retroviral infection with the IB constructs. The cells cannot be clonally selected because of the limitations in the length of time for which human endothelial cells can be passaged. Those cells that express relatively "high" levels of the wild-type protein and that may indeed have some reduced capacity to respond to NF-B activating agents may therefore not be representative of the population. Any changes in TNF-–induced ICAM-1 or E-selectin expression in that subpopulation of cells may not be apparent on analysis of the entire population.

Second, although retroviral expression of the full-length IB does indeed result in a reduction in NF-B DNA binding activity, the residual activity may be adequate to participate in TNF-–induced expression of ICAM-1 and E-selectin. The stability of the IBN protein, however, leads to a phenotypic effect even in those endothelial cells that express relatively small quantities of IBN. The presence of the truncated protein results in the formation of stable NF-B/IB complexes in the majority of the cells in the population. On treatment with TNF-, loss of NF-B–mediated ICAM-1 and E-selectin expression is therefore observed as a phenotypic characteristic of the population as a whole.

Those HUVECs expressing the IBN protein also appear to have reduced basal levels of ICAM-1 at the cell surface compared with control or IBwt-expressing cells. The HUVEC-IBN cells that were not treated with TNF- exhibited ICAM-1 values that were equivalent to the isotype-matched IgG1 negative control values (Fig 7A). The altered level of basal ICAM-1 expression in these cells may therefore indicate that NF-B, either directly or indirectly, also participates in the modulation of constitutive levels of ICAM-1.

Previous studies have demonstrated a crucial role for NF-B in mediating the cytokine-induced upregulation of adhesion molecule expression (E-selectin, ICAM-1, and VCAM-1).^{2 3 4 6 7 8 29} There have been no reports, however, of modulating NF-B DNA binding activity in endothelial cells using a methodology similar to ours. The use of retroviruses to express a proteolysis-resistant IB is designed to provide an endothelial cell population that is both lacking NF-BDNA binding activity and is stable over a period of time. Furthermore, retroviral transduction of human endothelial cells does not appear to affect the activation state of the cells compared with control uninfected HUVECs.

A study using an adenoviral vector to prevent NF-B activation in endothelial cells by overexpressing IB has been reported⁹ ; however, the use of adenoviral vectors for such a purpose may have several potential problems. That report did not demonstrate the levels of NF-B DNA binding activity in unstimulated control adenovirus-infected cells versus the uninfected endothelial cells, although it was indicated that adenovirus infection per se had an effect on IL-1 and IL-6 mRNA levels. It is possible that activation of inflammation-associated gene expression in the endothelial cells may occur by either direct and/or indirect effects of expression of adenoviral genes. The use of an adenoviral vector in and of itself may thereby lead to confounding results when using adenovirus-infected populations for studying endothelial cell activation and response to injury.

It has been shown that IB is capable of complexing with all NF-B family members that possess a transactivation domain^{11 12} ; thus, it provides a general target for using a dominant-negative strategy to prevent activation of NF-B. Furthermore, although agents such as antioxidants or protease and proteasome inhibitors are effective inhibitors of NF-B activation,^{2 19 30 31} they may exert broad-ranging effects that may also influence NF-B–mediated regulatory events via mechanisms that are not directly functioning in the NF-B activation pathway. Our approach of specifically preventing NF-B function through retroviral expression of the N-terminal IB deletion protein offers a high degree of specificity for investigating the role of NF-B in regulating endothelial adhesion molecule expressions. The pivotal role of NF-B in modulating cellular inflammatory responses and other key cellular processes such as apoptosis^{32 33 34} is becoming increasingly evident, and the generation of NF-B-minus human endothelial cells provides a useful tool for delineating the role of NF-B in regulating those processes in the endothelium.

	Selected Abbreviations and Acronyms

 

DTT = dithiothreitol FVIII-RA = factor VIII–related antigen ß-gal = ß-galactosidase HUVEC = human umbilical vein endothelial cell ICAM-1 = intercellular adhesion molecule-1 IL = interleukin N = N-terminal deletion NF-B = nuclear factor-B PMSF = phenylmethylsulfonyl fluoride TNF- = tumor necrosis factor- VCAM-1 = vascular cell adhesion molecule-1 wt (combination form) = wild-type

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-48925 (Dr Lockyer) and HL-34691 (Dr Buda) and a grant from the American Heart Association, Louisiana Affiliate, Inc (Dr Lockyer).

Received May 19, 1997; accepted November 18, 1997.

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