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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:1414.)
© 2001 American Heart Association, Inc.

Vascular Biology

Adenovirus-Mediated Overexpression of Dominant-Negative Mutant of c-Jun Prevents Intercellular Adhesion Molecule-1 Induction by LDL

A Critical Role for Activator Protein-1 in Endothelial Activation

Nanping Wang; Lynne Verna; Hai-ling Liao; Alex Ballard; Yi Zhu; Michael B. Stemerman

From the Division of Biomedical Sciences, University of California, Riverside.

Correspondence to Nanping Wang, MD, PhD, Division of Biomedical Sciences, University of California, Riverside, CA 92521. E-mail nwang{at}ucrac1.ucr.edu

	Abstract

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Abstract—— Low density lipoprotein (LDL) induces intercellular adhesion molecule-1 (ICAM-1) gene expression and leads to endothelial cell (EC) leukocyte adhesion. However, the transcriptional mechanism for LDL-induced EC perturbation remains to be fully explained. Activator protein-1 (AP-1) is induced after the exposure of ECs to LDL. In the present study, a regulated adenovirus expressing a dominant-negative mutant of c-Jun (TAM-67) was used to examine the role of AP-1 in the LDL-induced ICAM-1 activation. Overexpression of TAM-67 specifically inhibited AP-1 activation and prevented the LDL-activated surface expression of ICAM-1 protein in human umbilical vein ECs and human coronary artery ECs. Northern analyses and promoter transactivation assays indicated that this effect of TAM-67 was likely mediated through a suppression of the transcriptional regulation of the ICAM-1 gene. Functionally, TAM-67 attenuated leukocyte adherence to ECs in response to LDL. Furthermore, electrophoresis mobility shift assays and site-directed mutagenesis suggested that an AP-1–like motif in the promoter region of the human ICAM-1 gene was a critical cis element for LDL induction. These results, for the first time, provide evidence suggesting that AP-1 is a major regulatory mechanism leading to endothelial activation.

Key Words: LDL • endothelium • adhesion molecules • transcription factors • gene expression

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Low density lipoprotein is among the major risk factors and predictors for the development of atherosclerosis.¹ LDL perturbs endothelial function,² which is manifested as an increase in free radicals,³ a decrease in NO production,⁴ an increase in plasminogen activator inhibitor-1,⁵ and recruitment of monocytes⁶ via the induction of vascular adhesion molecules.^7–9 However, unlike agents such as inflammatory cytokines and endotoxins, native LDL does not appear to be a stimulator of nuclear factor-B (NF-B), a transcription factor¹⁰ that is thought to play a predominant role in endothelial cell (EC) activation. Rather, LDL induces other transcription factors, such as Egr-1¹¹and activator protein-1 (AP-1). In particular, LDL provokes a sustained elevation of c-Jun expression, AP-1 DNA binding, and transactivation activity in ECs.¹⁰ Moreover, the LDL induction of AP-1 is preceded by a rapid activation of c-Jun N-terminal kinase (JNK), the immediate upstream activator for c-Jun.¹² Recent observation in hypercholesterolemic rabbits revealed a persistent activation of JNK in atherosclerotic intima,¹³ indicating a possible link between AP-1 activation and atherogenesis.

Exposure to LDL increases endothelial expression of intercellular adhesion molecule-1 (ICAM-1), which is a counterreceptor for leukocyte function–associated antigen-1 and is a major component of inflammatory cell recruitment to ECs. Although extensive evidence has pointed to an important role of ICAM-1 in atherogenesis, the transcriptional mechanism by which LDL regulates this adhesion molecule has been largely unknown. Thus, the aim of the present study was to examine whether AP-1 is functionally important in ICAM-1 induction and the ensuing EC phenotypic activation in response to LDL. By using a tightly regulated adenovirus expressing a dominant-negative mutant of c-Jun, we have demonstrated that specific inhibition of AP-1 potently attenuated LDL-induced ICAM-1 expression and monocyte recruitment. Furthermore, an AP-1–like motif in the promoter region of the human ICAM-1 gene was identified as a critical cis element for LDL induction. These results, for the first time, provide evidence suggesting that AP-1 is a major regulatory mechanism leading to endothelial activation.

	Methods

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Cell Culture and LDL
Human umbilical vein ECs (HUVECs) were isolated from umbilical cords and maintained in EC medium containing 20% FBS, 20 mmol/L HEPES (pH 7.4), 5 ng/mL recombinant human fibroblast growth factor, 90 µg/mL heparin, and antibiotics.¹⁴ Human coronary artery ECs (HCAECs) were purchased from Clonetics, cultured in EC growth medium (EGM-2 MV, Clonetics) with 5% FBS, and changed to EC medium after confluence. THP-1, a human monocyte cell line (American Type Culture Collection), was grown in RPMI 1640 containing 10% FBS.¹⁵ LDL (1.019 g/mL<density<1.063 g/mL) was isolated from human plasma by a 2-step ultracentrifuge method as described previously.¹⁶

Adenoviral Vectors
To generate adenoviruses expressing the dominant-negative c-Jun mutant (AdTAM-67), the cDNA fragment encoding a truncated (residues 3 to 122) c-Jun (TAM-67, provided by M.J. Birrer, NCI, Rockville, MD)¹⁷ was subcloned into pAdlox and recombined with an E1- and E3-deleted adenovirus-5 genome DNA (5).^14,18 The expression of the inserted gene was driven by a 7 x tet operon/minimal cytomegalovirus promoter, which was further under the control of a tetracycline-responsive transactivator (tTA). The adenovirus expressing green fluorescence protein (AdGFP) was similarly constructed. The adenoviruses were plaque-purified and titrated in 293 cells as described.¹⁹

Electrophoretic Mobility Shift Assay
An electrophoretic mobility shift assay was performed as described previously.¹⁴ The sequences of oligonucleotides are as follows: (1) ICAM–AP-1a (-1290), 5'-TGG CCA GTG ACT CGC AGC CCC-'3; (2) ICAM–AP-1b (-940), 5'-GCT GCT GCC TCA GTT TCC CAG-3'; (3) ICAM–AP-1c (-321), 5'-TAG ACC GTG ATT CAA GCT TAG-3'; (4) ICAM–NF-B1 (-536), 5'-GCC CGG GGA GGA TTC CTG GGC CC-3'; and (5) ICAM–NF-B2 (-223), 5'-TTT AGC TTG GAA ATT CCG GAG CT-3'.

Plasmids and Transfection
The ICAM-1 promoters/reporters were constructed by subcloning progressively truncated segments of a 1344-bp 5' flanking region of the human ICAM-1 gene (provided by Dr T. Parks, Boehringer-Ingelheim Pharmaceuticals Inc, Ridgefield, Conn)²⁰ into pGL3-basic (Promega). The 7 x AP-1-Luc and 5 x NF-B-Luc are luciferase reporters containing 7 x AP-1 binding sites or 5 x -B sites (Stratagene). Plasmid expressing the yeast transcription factor Gal4 and 4 x UAS-TK-Luc have been previously reported (gifts from Barry Forman, City of Hope, Duarte, Calif).²¹ The plasmids were transfected into HUVECs by using a cationic lipid-based reagent (Targefect, Targeting System). Cell lysates were harvested to measure luciferase activity. In all transfection experiments, an RSV-ß-gal plasmid was cotransfected to normalize transfection efficiency.

Site-Directed Mutagenesis
Site-directed mutagenesis was performed by using the Quickchange kit (Stratagene) according to the manufacturer’s protocol. The mutagenic primers were synthesized to introduce mutations (lowercase letters) into the AP-1–like motif (underlined). The sequence of the primer (forward) is 5'-GCG GTG TAG ACC GTG gTg CcA GCT TAG CCT GGC CG-3' (the reverse primer was complementary to the forward primer). The mutated clones bearing the desired mutations were screened by restriction digestion (the mutation introduced an additional HindIII site) and confirmed by DNA sequencing.

Northern Blot Analysis
Total RNA was isolated by using Trizol reagent (GIBCO-BRL), fractionated, transferred to a nylon membrane, and hybridized to random-primed cDNA probes for the human ICAM-1 and the GAPDH genes.¹⁷

Western Blot Analysis
Protein samples were resolved on SDS-PAGE, transferred onto Immobilon-P membranes (Millipore), and reacted with rabbit polyclonal antibodies to c-Jun (sc-44, Santa Cruz) and a horseradish peroxidase–conjugated secondary antibody (sheep anti-rabbit, 1:5000, Sigma Chemical Co) followed by detection by enhanced chemiluminescence (ECL, Amersham).¹⁷

Immunofluorescence Staining
After fixation with 4% paraformaldehyde, HUVECs were permeabilized with 1% Triton X-100 and incubated with primary rabbit antisera for 1 hour. The cells were washed with PBS and then reacted with a fluorescein isothiocyanate–conjugated anti-rabbit antibody. Background fluorescence was determined by using the secondary antibody alone and subtracted. The results were observed with a fluorescence microscope.

Fluorescence-Linked Immunoassay of ICAM-1
HUVECs or HCAECs grown in 6-well plates were reacted with a mouse monoclonal antibody against ICAM-1 (Seratec). The secondary antibody was a fluorescein isothiocyanate–conjugated rabbit anti-mouse IgG. The fluorescence was measured with a fluorescence concentration analyzer (Pandex, IDEXX).⁹

Monocyte Adhesion Assay
HUVECs were grown to confluence on slides in a chamber (Lab-Tek) and incubated for 4 days with or without LDL. THP-1 cells were infected with AdGFP and AdtTA for 24 hours and then coincubated with the HUVECs for 30 minutes. The nonadhering cells were washed off, and the adhered THP-1 cells were observed and counted by use of a fluorescence microscope.

Statistical Analysis
Quantitative data were expressed as mean±SEM. Statistical analysis was performed with the Student t test. Differences were considered significant at P<0.05.¹⁷

	Results

Top Abstract Introduction Methods Results Discussion References

 
Regulated Expression of Dominant-Negative c-Jun in ECs
To directly assess the potential requirement for AP-1 in LDL-triggered EC signaling, we overexpressed a dominant-negative mutant of c-Jun, TAM-67, in ECs. Confluent HUVECs were coinfected with AdTAM-67 and AdtTA. At different time points after infection, cellular proteins were isolated and examined for TAM-67 expression by Western analysis with use of an antibody raised against the conserved DNA-binding domain of c-Jun (SC-44). As shown in Figure 1A, a truncated c-Jun protein (30 kDa) was readily detected 24 hours after infection. To regulate the TAM-67 expression, adenovirally infected ECs were maintained in medium containing varying concentrations of tetracycline (Tc, 01.0 µg/mL). Tc arrested the TAM-67 expression in a dose-dependent fashion. Endogenous wild-type c-Jun protein was also detected at a low level under basal conditions and was not suppressed by TAM-67. However, overexpression of TAM-67 appeared to decrease the induction of endogenous c-Jun by phorbol 12-myristate 13-acetate (PMA) and, to lesser extent, by LDL. Furthermore, immunofluorescence staining showed that after infection by AdTAM-67 and AdtTA, the mutant was detectable exclusively in the nuclei of ECs (Figure 1B) when stained with the c-Jun antibody SC-44 but not with the c-Jun antibody SC-45, which recognizes the N-terminal epitope (data not shown). In noninfected cells or the infected cells maintained in Tc medium, only the endogenous c-Jun was detected.

View larger version (46K): [in this window] [in a new window]   Figure 1. Regulated expression of the dominant-negative c-Jun protein in ECs. A, Western blot analysis. In the top left blot, cellular protein was isolated at indicated time points after infection with AdTAM-67 and AdtTA. c-Jun protein was detected by using an antibody against the DNA-binding domain of c-Jun (epitope corresponding to residues 247 to 263). In the bottom left blot, Tc tightly controls TAM-67 expression. Asterisk indicates endogenous c-Jun protein. In the right blot, HUVECs were infected with AdTAM-67 and maintained in the presence or absence of Tc. The cells were treated with LDL (240 mg/dL) or PMA (50 ng/mL) for 12 hours. B, Immunofluorescence staining of TAM-67. C, Overexpression of TAM-67 showing inhibition of AP-1 activation in HUVECs. Twelve hours after infection, HUVECs grown in 6-well plates were transfected with the reporter plasmid 7x AP-1-Luc, 5x NF-B-Luc, or 4x UAS-TK-Luc. The 4x UAS-TK-Luc was transfected together with either Gal4 as a positive control or blank pSG65 as a negative control. In all transient transfection experiments, the pRSV-Gal plasmid (0.2 µg per well) was cotransfected to normalize the transfection efficiency. Transfected cells were treated with LDL (240 mg/dL for 48 hours) or PMA (50 ng/mL for 16 hours) in the presence or the absence of Tc. In all LDL incubation experiments, the medium was changed daily unless otherwise described. The luciferase activity was measured and normalized to ß-galactosidase activity. The results were expressed as the fold induction relative to untreated basal controls with Tc. The data shown are from 3 experiments performed in triplicate. *P<0.05 and **P<0.01 for comparison of reporter activity with or without Tc.

Inhibition of AP-1 by Overexpression of TAM-67 in ECs
Transient transfection assays with the AP-1–dependent luciferase reporter plasmid, 7 x AP-1-Luc, were carried out to determine the functional efficacy of TAM-67 for suppressing AP-1 activity in endothelial cells. HUVECs were infected with AdTAM-67 plus AdtTA and then transfected with 7 x AP-1-Luc (Figure 1C). The transfected ECs were then treated with LDL for 48 hours and measured for luciferase activity. The results showed that TAM-67 suppressed LDL-induced AP-1 activation. Furthermore, luciferase reporters for NF-B (5 x NF-B-Luc) and for the yeast transcription factor Gal4 (4 x UAS-TK-Luc) were used to evaluate the effect of the dominant-negative c-Jun on other specific or general transcriptional mechanisms. As shown in Figure 1C, the NF-B and the Gal4-dependent reporters were activated by exposure to PMA and overexpression of Gal4, respectively. Basal activity and induced activity of these transcription factors were unaffected by TAM-67 at the same level at which it significantly suppresses AP-1 activity.

Blocking AP-1 Inhibited LDL Induction of ICAM-1 in ECs
To examine whether inhibition of AP-1 could prevent ICAM-1 induction by LDL, confluent HUVECs were infected with AdTAM-67 and AdtTA and, thereafter, maintained in medium (containing 20% FBS) with or without Tc. After infection, the cells were exposed to LDL for 4 days. Total RNA was isolated and hybridized for ICAM-1 transcripts. As shown in Figure 2A, the ICAM-1 mRNA expression induced by LDL was silenced by TAM-67, whereas the expression of GAPDH, a housekeeping gene, was not affected. Subsequently, promoter transactivation assays were performed to examine whether a transcriptional mechanism accounted for the inhibitory effect of TAM-67. As shown in Figure 2B, LDL induction of ICAM-1 promoter activity was markedly inhibited by TAM-67, indicating that TAM-67 decreases LDL-induced ICAM-1 mRNA by suppressing the promoter activation. Next, surface expression of ICAM-1 was examined by immunofluorescence to determine whether the suppressive effect also occurs at the protein level. Interestingly, in HUVECs and HCAECs, the ICAM-1 protein level was increased after exposure to LDL, and the induction was diminished by overexpression of TAM-67 (Figure 2C). Thus, it is suggested that the AP-1 may be an important regulator in LDL-induced ICAM-1 expression.

View larger version (27K): [in this window] [in a new window]   Figure 2. TAM-67 inhibits LDL-induced ICAM-1 expression. A, Northern analysis. Control or AdTAM-67–infected cells were incubated with LDL (240 mg/dL) for 4 days. Total RNA was isolated and hybridized with a 32P-labeled cDNA probe for human ICAM-1 or GAPDH. B, Promoter transactivation assay. The infected cells were transfected with an ICAM-1 promoter/reporter, -455-Luc (1.8 µg per well), and pRSV-Gal (0.2 µg per well) and then exposed to LDL for 48 hours. The results were expressed as the fold induction relative to untreated basal control with Tc. C, Surface expression of ICAM-1. HUVECs or HCAECs were infected with AdTAM-67 and exposed to LDL or control medium for 4 days in the presence or absence of Tc. Surface expression of ICAM-1 was detected by indirect immunofluorescence. Fluorescence intensity was measured and expressed as the mean±SEM. *P<0.05 for comparison of treatment under conditions with or without Tc.

Inhibition of AP-1 Attenuates LDL-Induced EC Monocyte Adhesion
To examine whether inactivation of AP-1 in ECs functionally modulates EC-monocyte interaction in response to LDL, ECs were coinfected with AdTAM-67 and AdtTA, in the presence or absence of Tc, and exposed to LDL. THP-1 cells were preinfected with AdGFP and AdtTA for 24 hours for direct visualization by fluorescence microscopy. These cells were incubated with LDL-exposed ECs for 30 minutes. As shown in Figure 3, LDL caused an 3-fold increase in monocyte binding to ECs. Overexpression of TAM-67 had no effect on the basal level of monocyte adherence but significantly decreased the LDL-induced EC-monocyte interaction. Therefore, inhibition of AP-1 activity in ECs could functionally attenuate the proinflammatory effects of LDL.

View larger version (18K): [in this window] [in a new window]   Figure 3. Inhibition of AP-1 attenuates LDL-induced EC-monocyte adhesion. The infected cells were exposed to control medium (A, a and b) or LDL (240 mg/dL; A, c and d) for 4 days with (A, a and c) or without (A, b and d) Tc. The EC-bound GFP-expressing THP-1 cells (green cells) were visualized under a fluorescence microscope (A). The EC-bound monocytes were counted and averaged from 3 fields per treatment. Results are expressed as fold increase relative to non–LDL-treated control without TAM-67 expression (A, a) and are representative of 3 experiments (B). *P<0.05 for comparison of monocyte adhesion with or without Tc.

LDL Induction of Human ICAM-1 Promoter
Structural analysis of the human ICAM-1 gene has revealed multiple AP-1–like and NF-B–like motifs within a 1.3-kb 5' flanking region (Figure 4). To delineate the promoter region(s) responsible for LDL induction, we transfected HUVECs with reporters fused to a series of 5' deletion mutants of the 1.3-kb fragment and exposed the cells to LDL. LDL caused a moderate induction of the 1.3-kb ICAM-1 promoter/reporter. However, deletion of the distal segment (-1300 to -942) decreased the basal activity but increased the promoter responsiveness to LDL. Further deletion of a segment containing an AP-1/Ets repeat (AP-1b) and a consensus NF-B site (ICAM-B1) caused little decrease in either basal activation or the LDL responsiveness. The most critical cis element(s) appears to reside in the 168-bp region (-445 to -277). Thus, it is suggested that the proximal AP-1 site (AP-1c) may be a candidate element to mediate the promoter responsiveness to LDL.

View larger version (12K): [in this window] [in a new window]   Figure 4. Induction of the promoter deletion constructs of human ICAM-1 gene by LDL. Putative transcription factor binding motifs were found on -1353 ICAM-Luc (the position is in relation to the transcription start site). HUVECs were transfected with the individual ICAM-1 promoters/reporters and then exposed to LDL for 48 hours. The data are from 3 independent experiments and expressed as mean±SEM of the ß-galactosidase normalized luciferase activity. *P<0.05 and **P<0.01 for comparisons between promoter activity with and without LDL exposure.

Proximal AP-1–Like Motif Is Required for LDL Induction
To examine whether LDL has an effect on the several putative binding motifs for AP-1 and NF-B transcription factors in the human ICAM-1 promoter, an electrophoretic mobility shift assay was carried out with oligonucleotide containing the sequences corresponding to the specific AP-1 or NF-B sites (Figure 5). LDL enhanced protein binding to the probe containing the proximal AP-1 sequence (AP-1c), caused moderate binding to the oligomers containing the AP-1/Ets repeats (AP-1b), and caused little binding to the oligomers containing the distal AP-1 (AP-1a). However, LDL had no detectable effect on the nuclear protein binding to oligomers for either of the NF-B sites. Specificity of the DNA binding activity was confirmed by using a 100-fold molar excess of the cold oligonucleotides to compete out the retarded bands (data not shown).

View larger version (38K): [in this window] [in a new window]   Figure 5. LDL induces DNA binding activity to AP-1 sites in the ICAM-1 promoter. Nuclear proteins extracted from HUVECs in (1) control, (2) LDL (240 mg/dL, 48 hours), or (3) PMA (100 ng/mL, 2 hours) groups were reacted with 32P-labeled oligonucleotides corresponding to AP-1–like or NF-B–like sequences in the ICAM-1 promoter. Arrows indicate specifically shifted bands for each probe as confirmed by cold competition experiments.

To specifically examine the functional contribution of the proximal AP-1 site to LDL-mediated transcriptional activation of ICAM-1, a mutant construct was generated to disrupt the proximal AP-1 site (-445mut). As shown in Figure 6, site-directed mutagenesis of the proximal AP-1 motif largely abolished the promoter responsiveness to LDL. Thus, the proximal AP-1–like motif appears to be a critical LDL-responsive element in the ICAM-1 promoter.

View larger version (12K): [in this window] [in a new window]   Figure 6. Site-directed mutagenesis of AP-1 motif abrogates LDL responsiveness. HUVECs were transfected with the wild-type (-445wt) or mutant (-445 mut) ICAM-1 promoters/reporters and exposed to LDL for 48 hours. The results were expressed as fold induction of the wild-type promoter under basal conditions and were representative of 4 experiments. *P<0.05 and **P<0.01 for comparisons between promoter activity of wild-type and mutant promoters under conditions of basal or LDL exposure.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
LDL is considered to be the main atherogenic class of lipoproteins, and an elevated level of LDL is one of the most important risk factors for atherosclerosis and cardiovascular morbidity. Increased levels of plasma LDL cholesterol are correlated with increased risk for complications of atherosclerosis, whereas lipid-lowering treatment can reduce coronary events and total mortality.¹ Continuous interaction between plasma LDL and arterial endothelium seems to be one of the factors pertinent to the development of atherosclerosis.² Although growing evidence supports the idea that oxidization of LDL may increase its ability to damage endothelial function, there is also an increasing body of evidence showing that a pathophysiological level of nonoxidized native LDL causes endothelial perturbations. In addition to our previous finding that LDL induces the expression of a number of adhesion molecules and increases proinflammatory cell adhesion to HUVECs, recent studies from our laboratory and other groups have largely expanded the ever-growing spectrum of atherogenic effects of native LDL.^4,7,11,22 Together, these observations argue for a pathophysiological role of LDL in the development of atherosclerosis and thrombosis. In search of the molecular mechanisms by which LDL may modulate the endothelial phenotype, we further demonstrate in the present study that (1) LDL-induced ICAM-1 expression and resultant EC-leukocyte adhesion can be attenuated by blocking AP-1 activation with adenovirus-mediated expression of a dominant-negative c-Jun in ECs, and (2) the AP-1–like motif in the ICAM-1 promoter appears to be a major cis element responsible for LDL induction. The present study shows, for the first time, that the AP-1 signaling pathway and its cognate binding motif are of major importance for endothelial activation by LDL.

AP-1 is a family of transcription factors composed of various dimers of Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra-1, and Fra-2). Family members form dimers and bind to DNA containing a TPA (12-O-tetradecanoyl-phorbol-13-acetate)-responsive element site to regulate gene expression. AP-1 provides pivotal regulatory roles coupling extracellular signals to key gene-activating events.²³ Deregulation of AP-1 is seen in ECs in response to a variety of pathological stimuli. Particularly, LDL induces a sustained activation of AP-1 in ECs. In contrast to proinflammatory cytokines, LDL lacks an effect on NF-B. Using adenovirus-mediated overexpression of c-jun and c-fos, we demonstrated that experimental activation of AP-1 is sufficient to cause endothelial activation, independent of NF-B.¹⁴ The present report further shows that specific inactivation of AP-1 diminished ICAM-1 expression and the ensuing leukocyte recruitment, indicating that LDL, as a chronic vascular agonist, perturbs the EC phenotype through a mechanism that is distinct from an acutely activated pathway.

ICAM-1 is an inducible cell adhesion glycoprotein expressed on the surface of a variety of cell types, including ECs, and plays an important role in leukocyte adhesion and inflammation.²⁴ It is also implicated in atherosclerosis.²⁵ Thus, elucidation of the transcriptional control of this molecule is important for understanding the inflammatory response in general as well as the pathogenesis and therapeutic intervention of atherosclerosis. In ECs, ICAM-1 is upregulated in response to a wide variety of stimuli, including proinflammatory cytokines, lipopolysaccharides, PMA, and H₂O₂. These stimuli increase ICAM-1 expression primarily through the activation of ICAM-1 gene transcription.²⁶ On the other hand, various species of lipoproteins, including LDL, oxidized LDL,²⁷ Lp(a),²⁸ and remnant lipoproteins,²⁹ have been reported to induce the expression of ICAM-1 in ECs. However, to our knowledge, there has been no preceding report identifying the regulatory elements responsible for ICAM-1 induction by these lipoproteins. Analysis of the 1.3-kb human ICAM-1 promoter region revealed multiple binding motifs for various transcription activators, including AP-1, NF-B, AP-2, Ets, signal transducers and activators of transcription, and SP-1.²⁶ Such a complex promoter structure appears to mediate gene induction in response to diverse environmental stimuli. In the present study, we provide initial evidence showing the proximal AP-1–like motif to be an intrinsic cis element responsible for ICAM-1 induction by LDL. The distal AP-1 appears not to be involved in LDL induction of ICAM-1 because LDL did not induce binding to this site. LDL induced moderate binding to the AP-1/Ets repeats, which have been previously identified as being required for induction by H₂O₂.³⁰ However, deletion of the region containing this motif did not cause marked reduction of the LDL response, suggesting that it is not a critical element for LDL. Although NF-B has been reported to be essential for ICAM-1 expression, we have recently demonstrated the induction of ICAM-1 and monocyte chemotactic protein-1 independent of NF-B activation.¹⁴ LDL does not activate the NF-B pathway and has no detectable effect on the protein binding to the oligomers containing a consensus NF-B motif.¹⁰ Similarly, LDL treatment appears to have little effect on the nuclear protein binding to the ICAM-1–specific NF-B sequences. Thus, it is likely that LDL activates ICAM-1 predominantly through an AP-1–mediated mechanism. Interestingly, such a stimulus-specific NF-B–independent mechanism is also reported in the induction of the interleukin-8 gene.³¹

Because AP-1–like motifs have been identified as recurrent regulatory sequences in a series of genes that are known to be important in inflammation, atherogenesis, and thrombogenesis, such as ICAM-1, vascular cell adhesion molecule-1, monocyte chemotactic protein-1, endothelin-1, tissue factor, and plasminogen activator inhibitor-1,^32–35 manipulation of this transcription factor is of particular significance for understanding vascular biology and therapeutic intervention as well. Currently, chemical antioxidants curcumin and N-acetylcysteine are frequently used to inhibit AP-1 activity. However, these chemicals also are reported to nonspecifically inhibit other pathways.^36,37 Although antisense oligonucleotides for c-jun or c-fos can be a more specific approach, low transfection efficiency and high cytotoxicities often limit their application. Thus, a dominant-negative approach was used in the present study to test the functional role of the AP-1 pathway. To disable all AP-1–containing transcription factors in ECs, an adenovirus was generated to express TAM-67, a well-characterized trans-dominant–negative mutant of c-Jun.^38,39 It retains the leucine zipper domain necessary for dimerization with Jun and Fos family members and the DNA binding domain, but the N-terminal region responsible for transcriptional activation has been deleted. TAM-67 may inhibit AP-1 activity by dimerization and direct competition with endogenous AP-1 for DNA binding, thus, quenching their transactivation activity. Additionally, suppression of endogenous c-Jun induction may interrupt the c-Jun autoregulatory loop. Regarding specificity, TAM-67 has been demonstrated to specifically block AP-1–dependent, but not NF-B–dependent, transcription.⁴⁰ In the present study, adenovirus-mediated overexpression of TAM-67 specifically suppressed AP-1 activity in ECs and, under the same conditions, had little effect on other transcription factors, such as NF-B and Gal4. Furthermore, using a tetracycline-regulated adenoviral system, we were able to manipulate AP-1 activity in an efficient and tightly controllable fashion in primary vascular ECs. This approach may have wide application for dissecting the specific role of AP-1 in various physiological and pathological conditions.

In conclusion, the present results demonstrate that AP-1 activation plays a critical role in endothelial phenotypic perturbation by LDL, which may represent a novel mechanism of endothelial response to LDL cholesterol. It is hoped that this finding may help in understanding the roles of LDL and transcription factors in vascular inflammatory processes and atherogenesis.

	Acknowledgments

 
This study was supported by National Institutes of Health grant HL-33742-15A1 (to M.B.S) and American Heart Association Scientist Development Grant 0130276N (to N.W.). We thank John Shyy for critical reading of this manuscript and Yue Wang for technical assistance with isolation of LDL and HUVECs.

Received June 12, 2001; accepted July 2, 2001.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Berenson GS, Srinivasan SR, Bao W, Newman WP III, Tracy RE, Wattigney WA. Association between multiple cardiovascular risk factors and atherosclerosis in children and young adults: the Bogalusa Heart Study. N Engl J Med. . 1998; 338: 1650–1656.[Abstract/Free Full Text]

2. Stemerman MB. Lipoprotein effects on the vessel wall. Circ Res. . 2000; 86: 715–716.[Free Full Text]

3. Pritchard KA Jr, Groszek L, Smalley DM, Sessa WC, Wu M, Villalon P, Wolin MS, Stemerman MB. Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion. Circ Res. . 1995; 77: 510–518.[Abstract/Free Full Text]

4. Feron O, Dessy C, Moniotte S, Desager JP, Balligand JL. Hypercholesterolemia decreases nitric oxide production by promoting the interaction of caveolin and endothelial nitric oxide synthase. J Clin Invest. . 1999; 103: 897–905.[Medline] [Order article via Infotrieve]

5. Ren S, Shatadal S, Shen GX. Protein kinase C-beta mediates lipoprotein-induced generation of PAI-1 from vascular endothelial cells. Am J Physiol Endocrinol Metab. . 2000; 278: E656–E662.[Abstract/Free Full Text]

6. Pritchard KA Jr, Tota RR, Lin JH, Danishefsky KJ, Kurilla BA, Holland JA, Stemerman MB. Native low density lipoprotein: endothelial cell recruitment of mononuclear cells. Arterioscler Thromb. . 1991; 11: 1175–1181.[Abstract/Free Full Text]

7. Allen S, Khan S, Al Mohanna F, Batten P, Yacoub M. Native low density lipoprotein-induced calcium transients trigger VCAM-1 and E-selectin expression in cultured human vascular endothelial cells. J Clin Invest. . 1998; 101: 1064–1075.[Medline] [Order article via Infotrieve]

8. Lin JH, Zhu Y, Liao HL, Kobari Y, Groszek L, Stemerman MB. Induction of vascular cell adhesion molecule-1 by low-density lipoprotein. Atherosclerosis. . 1996; 127: 185–194.[Medline] [Order article via Infotrieve]

9. Smalley DM, Lin JH, Curtis ML, Kobari Y, Stemerman MB, Pritchard KA Jr. Native LDL increases endothelial cell adhesiveness by inducing intercellular adhesion molecule-1. Arterioscler Thromb Vasc Biol. . 1996; 16: 585–590.[Abstract/Free Full Text]

10. Zhu Y, Lin JH, Liao HL, Friedli O Jr, Verna L, Marten NW, Straus DS, Stemerman MB. LDL induces transcription factor activator protein-1 in human endothelial cells. Arterioscler Thromb Vasc Biol. . 1998; 18: 473–480.[Abstract/Free Full Text]

11. Cui MZ, Penn MS, Chisolm GM. Native and oxidized low density lipoprotein induction of tissue factor gene expression in smooth muscle cells is mediated by both Egr-1 and Sp1. J Biol Chem. . 1999; 274: 32795–32802.[Abstract/Free Full Text]

12. Zhu Y, Liao HL, Wang N, Friedli O Jr, Verna L, Stemerman MB. Low-density lipoprotein activates Jun N-terminal kinase (JNK) in human endothelial cells. Biochim Biophys Acta. . 1999; 1436: 557–564.[Medline] [Order article via Infotrieve]

13. Metzler B, Hu Y, Dietrich H, Xu Q. Increased expression and activation of stress-activated protein kinases/c-Jun NH(2)-terminal protein kinases in atherosclerotic lesions coincide with p53. Am J Pathol. . 2000; 156: 1875–1886.[Abstract/Free Full Text]

14. Wang N, Verna L, Hardy S, Forsayeth J, Zhu Y, Stemerman MB. Adenovirus-mediated overexpression of c-Jun and c-Fos induces intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 in human endothelial cells. Arterioscler Thromb Vasc Biol. . 1999; 19: 2078–2084.[Abstract/Free Full Text]

15. Tsuchiya S, Yamabe M, Yamaguchi Y, Kobayashi Y, Konno T, Tada K. Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int J Cancer. . 1980; 26: 171–176.[Medline] [Order article via Infotrieve]

16. Pritchard KA Jr, Holland JA, Rogers NJ, Crean CC, Britton TE, Onigman P, Stemerman MB. Low-density lipoprotein preparation by combined diafiltration and ultracentrifugation. Anal Biochem. . 1988; 174: 121–127.[Medline] [Order article via Infotrieve]

17. Wang N, Verna L, Hardy S, Zhu Y, Ma KS, Birrer MJ, Stemerman MB. c-Jun triggers apoptosis in human vascular endothelial cells. Circ Res. . 1999; 85: 387–393.[Abstract/Free Full Text]

18. Hardy S, Kitamura M, Harris-Stansil T, Dai Y, Phipps ML. Construction of adenovirus vectors through Cre-lox recombination. J Virol. . 1997; 71: 1842–1849.[Abstract/Free Full Text]

19. Graham FL, Prevec L. Methods for construction of adenovirus vectors. Mol Biotechnol. . 1995; 3: 207–220.[Medline] [Order article via Infotrieve]

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

21. Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, Evans RM. 15-Deoxy-delta 12, 14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR gamma. Cell. . 1995; 83: 803–812.[Medline] [Order article via Infotrieve]

22. Sachinidis A, Kettenhofen R, Seewald S, Gouni-Berthold I, Schmitz U, Seul C, Ko Y, Vetter H. Evidence that lipoproteins are carriers of bioactive factors. Arterioscler Thromb Vasc Biol. . 1999; 19: 2412–2421.[Abstract/Free Full Text]

23. Karin M, Liu Z, Zandi E. AP-1 function and regulation. Curr Opin Cell Biol. . 1997; 9: 240–246.[Medline] [Order article via Infotrieve]

24. van de SA, van der Saag PT. Intercellular adhesion molecule-1. J Mol Med. . 1996; 74: 13–33.[Medline] [Order article via Infotrieve]

25. Cybulsky MI, Lichtman AH, Hajra L, Iiyama K. Leukocyte adhesion molecules in atherogenesis. Clin Chim Acta. . 1999; 286: 207–218.[Medline] [Order article via Infotrieve]

26. Collins T, Read MA, Neish AS, Whitley MZ, Thanos D, Maniatis T. Transcriptional regulation of endothelial cell adhesion molecules: NF-kappa B and cytokine-inducible enhancers. FASEB J. . 1995; 9: 899–909.[Abstract]

27. Takei A, Huang Y, Lopes-Virella MF. Expression of adhesion molecules by human endothelial cells exposed to oxidized low density lipoprotein: influences of degree of oxidation and location of oxidized LDL. Atherosclerosis. . 2001; 154: 79–86.[Medline] [Order article via Infotrieve]

28. Takami S, Yamashita S, Kihara S, Ishigami M, Takemura K, Kume N, Kita T, Matsuzawa Y. Lipoprotein(a) enhances the expression of intercellular adhesion molecule-1 in cultured human umbilical vein endothelial cells. Circulation. . 1998; 97: 721–728.[Abstract/Free Full Text]

29. Doi H, Kugiyama K, Oka H, Sugiyama S, Ogata N, Koide SI, Nakamura SI, Yasue H. Remnant lipoproteins induce proatherothrombogenic molecules in endothelial cells through a redox-sensitive mechanism. Circulation. . 2000; 102: 670–676.[Abstract/Free Full Text]

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

31. Roebuck KA, Carpenter LR, Lakshminarayanan V, Page SM, Moy JN, Thomas LL. Stimulus-specific regulation of chemokine expression involves differential activation of the redox-responsive transcription factors AP-1 and NF-kappaB. J Leukoc Biol. . 1999; 65: 291–298.[Abstract]

32. Lee ME, Dhadly MS, Temizer DH, Clifford JA, Yoshizumi M, Quertermous T. Regulation of endothelin-1 gene expression by Fos and Jun. J Biol Chem. . 1991; 266: 19034–19039.[Abstract/Free Full Text]

33. Olman MA, Hagood JS, Simmons WL, Fuller GM, Vinson C, White KE. Fibrin fragment induction of plasminogen activator inhibitor transcription is mediated by activator protein-1 through a highly conserved element. Blood. . 1999; 94: 2029–2038.[Abstract/Free Full Text]

34. Parry GC, Mackman N. Transcriptional regulation of tissue factor expression in human endothelial cells. Arterioscler Thromb Vasc Biol. . 1995; 15: 612–621.[Abstract/Free Full Text]

35. Shyy JY, Lin MC, Han J, Lu Y, Petrime M, Chien S. The cis-acting phorbol ester "12-O-tetradecanoylphorbol 13-acetate"-responsive element is involved in shear stress-induced monocyte chemotactic protein 1 gene expression. Proc Natl Acad Sci U S A. . 1995; 92: 8069–8073.[Abstract/Free Full Text]

36. Bouloumie A, Marumo T, Lafontan M, Busse R. Leptin induces oxidative stress in human endothelial cells. FASEB J. . 1999; 13: 1231–1238.[Abstract/Free Full Text]

37. Pendurthi UR, Williams JT, Rao LV. Inhibition of tissue factor gene activation in cultured endothelial cells by curcumin: suppression of activation of transcription factors Egr-1, AP-1, and NF-kappa B. Arterioscler Thromb Vasc Biol. . 1997; 17: 3406–3413.[Abstract/Free Full Text]

38. Alani R, Brown P, Binetruy B, Dosaka H, Rosenberg RK, Angel P, Karin M, Birrer MJ. The transactivating domain of the c-Jun proto-oncoprotein is required for cotransformation of rat embryo cells. Mol Cell Biol. . 1991; 11: 6286–6295.[Abstract/Free Full Text]

39. Brown PH, Chen TK, Birrer MJ. Mechanism of action of a dominant-negative mutant of c-Jun. Oncogene. . 1994; 9: 791–799.[Medline] [Order article via Infotrieve]

40. Petrak D, Memon SA, Birrer MJ, Ashwell JD, Zacharchuk CM. Dominant negative mutant of c-Jun inhibits NF-AT transcriptional activity and prevents IL-2 gene transcription. J Immunol. . 1994; 153: 2046–2051.[Abstract]

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