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(Circulation Research. 1999;85:387-393.)
© 1999 American Heart Association, Inc.

Molecular Medicine

c-Jun Triggers Apoptosis in Human Vascular Endothelial Cells

Nanping Wang, Lynne Verna, Stephen Hardy, Yi Zhu, Kuo-Sheng Ma, Michael J. Birrer, Michael B. Stemerman

From the Division of Biomedical Sciences (N.W., L.V., Y.Z., K.-S.M., M.B.S.), University of California, Riverside, Calif; Chiron Corporation (S.H.), Emeryville, Calif; and Medicine Branch (M.J.B.), National Cancer Institute, National Institutes of Health, Rockville, Md.

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

	Abstract

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Abstract—In endothelial cells (ECs), the transcription factor c-Jun is induced by a variety of stimuli that perturb EC function. To extend our understanding of the role of c-Jun in EC physiology, we have directed overexpression of c-Jun in human umbilical vein ECs by using a tetracycline-regulated adenoviral expression system. In this study, we report a novel observation using this system. Specific expression of c-Jun is a sufficient trigger for ECs to undergo apoptosis, as demonstrated by a set of combined assays including an ELISA specific for histone-associated DNA fragmentation, DNA laddering, and TdT-mediated dUTP nick end labeling (TUNEL). Tetracycline can effectively shut off c-Jun overexpression and prevent EC apoptosis. Cleavage of poly(ADP-ribose) polymerase was also detected in ECs overexpressing c-Jun. Moreover, inhibitors of cysteine proteases blocked the apoptosis, suggesting a caspase-associated mechanism involved in proapoptotic effects of c-Jun. To gain further insight into the role of c-Jun as a pathophysiological regulator of EC death, TAM67, a dominant-negative mutant of c-Jun, was overexpressed in human umbilical vein ECs to abrogate endogenous c-Jun/activator protein-1 activation. H₂O₂-triggered apoptosis was largely attenuated in ECs overexpressing TAM67. Together, these results suggest that c-Jun, as a proapoptotic molecule, may play a role in mediating the cell death program in vascular endothelium.

Key Words: c-Jun • adenovirus • endothelial cell • apoptosis • hydrogen peroxide

	Introduction

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Vascular endothelium, when unperturbed, is considered to provide a relatively nonadhesive and nonthrombotic interface. This characteristic is likely essential to physiological homeostasis. Endothelial cell (EC) apoptosis has been implicated in numerous pathophysiological processes, such as angiogenesis, thrombosis, and atherosclerosis.¹ The molecular mechanisms controlling EC apoptosis and, particularly, its transcriptional regulation remain largely unknown. Recently, the transcription factor c-Jun has been shown to have diverse roles in the apoptotic process, depending on cell type and microenvironment. Involvement of the c-jun gene in EC apoptosis has been implied by observations showing that many EC-perturbing agents, such as inflammatory cytokines,² lipopolysaccharides,³ reactive oxygen,⁴ and oxidized LDL,⁵ induce EC apoptosis as well as c-jun expression. Because these stimuli usually have pleiotropic effects on signal transduction pathways, it is not clear whether induction of c-jun in ECs is causally related to evoking apoptosis or, instead, is protective. Indeed, c-jun may even be an innocent bystander in EC apoptosis. To clarify this potentially important control mechanism, we have used a tetracycline-regulated adenoviral expression system for c-jun. The expression of c-jun can be exquisitely controlled using this process, and the findings of its expression are reported here. In addition, to gain further insight into the role of endogenous c-Jun as a pathophysiological regulator of EC death, TAM67, a dominant-negative mutant of c-Jun, was overexpressed in human umbilical vein ECs (HUVECs), and its effects on H₂O₂–triggered apoptosis were examined. These results indicate a key role for c-jun as a mediator of EC apoptosis.

	Materials and Methods

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Adenoviruses and Gene Transfer
To construct the recombinant adenovirus expressing the human c-jun gene (AdJun), a cDNA fragment containing the full-length coding regions was obtained from the parental plasmid pRSV-jun (generous gift from Dr R. Tjian, University of California, Berkeley).⁶ It was subcloned into a shuttle plasmid, pAdlox, and recombined with an E1- and E3-deleted 5 viral DNA in CRE8 cells. Expression of the inserted gene was driven by a 7x tet/minimal cytomegalovirus promoter that was further under the control of an artificial tetracycline-responsive transactivator (tTA).⁷ Adenoviruses expressing the ß-galactosidase gene (Adß-gal) and the tTA gene (AdtTA) were constructed from pUHC-13-3 and pUHD 15-1, as described previously.⁸ Dominant-negative c-Jun (TAM67) was generated from wild-type c-Jun by deletion of residues 3 to 122 in the amino-terminal transcriptional activation domain.⁹ The recombinant adenovirus expressing dominant-negative c-Jun mutant (AdTAM67) was generated as described.¹⁰ The adenoviruses were plaque-purified, expanded and titrated in 293 cells, and purified by cesium chloride methods.¹¹

For adenovirus-mediated gene transfer, confluent HUVECs were exposed to adenoviral vectors at a multiplicity of infection (MOI) of 100–200 for 2 hours (AdtTA was coinfected with AdJun or Adß-gal to induce the tetracycline-controllable expression). After the viruses were washed off, infected cells were further incubated for the indicated time courses in the presence or absence of tetracycline.

Cell Culture and Reagents
HUVECs were harvested by collagenase treatment of umbilical cord veins and cultured on plates coated with collagen.¹² Cells were maintained in M199 supplemented with 20% FBS, 20 mmol/L HEPES (pH 7.4), 1 ng/mL recombinant human fibroblast growth factor, 90 µg/mL heparin, and antibiotics. In all of the experiments, cells within 3 passages were used. Hydrogen peroxide (H₂O₂), DAPI, and MTT were purchased from Sigma. The peptide inhibitors zDEVD.fmk and zVAD.fmk were from Calbiochem.

Assessment of Apoptosis
Detection of DNA Fragmentation by ELISA
Quantitative analysis of DNA fragmentation was carried out using a histone-based ELISA system (Boehringer Mannheim). HUVECs in 6-well plates were incubated for 36 hours after infection with AdJun in the presence or absence of tetracycline (0.1 µg/mL) and then lysed. The histone-associated DNA fragments were linked to a mouse anti-histone antibody, and the DNA part of the nucleosome was linked to the anti–DNA-peroxidase. The amount of peroxidase retained in the immunocomplex was photometrically determined.

DNA Laddering
After treatment, attached cells and floating cells were combined and lysed in 0.2 mL of lysis buffer (4 mol/L urea, 100 mmol/L Tris, 20 mmol/L NaCl, and 200 mmol/L EDTA [pH 7.4]) and 40 µL of proteinase K solution (20 mg/mL in 50 mmol/L Tris-HCl [pH 8.0] and 1 mmol/L CaCl₂) for 1 hour at 55°C. After elution and isopropanol precipitation, DNA was resuspended in Tris-EDTA buffer, fractionated on 1.5% agarose gel in 1x Tris-boric acid-EDTA buffer, and stained with ethidium bromide.

DAPI Staining
For morphological evaluation of nuclei, cells were stained with DAPI solution (0.2% µg/mL) for 20 minutes and then visualized by fluorescence microscopy.

TdT-Mediated dUTP Nick End-Labeling (TUNEL) Assay
A TUNEL kit was used according to the manufacturer's instruction (Oncor). Confluent HUVECs were infected with AdJun or with Adß-gal at the same MOI and seeded in chamber slides (Lab-Tek) in the presence or absence of tetracycline (0.1 µg/mL) for 36 hours. Cells were fixed with 1% paraformaldehyde. The 3'-nick ends were labeled with digoxigenin-dUTP and a fluorescein-conjugated anti-digoxigenin antibody. Propidium iodide was used to counterstain the nuclei.

Determination of Cell Viability
Cell viability was measured by means of the MTT assay. HUVECs were grown to confluence in 96-well plates and were exposed to apoptotic stimuli or control medium for 18 hours. Cells were incubated with MTT solution (0.5 mg/mL) for 4 hours at 37°C, medium was removed, and cells were lysed with 2-isopropanol containing 0.04 mol/L HCl. The metabolized MTT was determined photometrically at 570 nm, with 690 nm as reference.

RNA Isolation and Northern Blot Analysis
Total RNA was isolated using Trizol reagent (Life Technologies), fractionated on a formaldehyde/agarose gel, transferred to a nylon membrane, and hybridized to random-primed cDNA probes for human c-jun and Von Willebrand factor (VWF) genes.¹²

Protein Isolation and Western Blot Analysis
Nuclear proteins were extracted from HUVECs as previously described.¹² Protein concentration was measured with the BCA protein assay kit (Pierce). Protein samples were resolved on SDS-PAGE, transferred onto Immobilon-P membrane (Millipore), and analyzed with rabbit polyclonal antibodies to c-Jun (N, catalog No. sc-45, Santa Cruz), JNK1 (FL, catalog No. sc-571, Santa Cruz), or poly(ADP-ribose) polymerase (PARP; Boehringer Mannheim) and a horseradish peroxidase–conjugated secondary antibody (sheep anti-rabbit, 1:5000, Sigma) followed by enhanced chemiluminescence detection (Amersham).

Statistical Analysis
Quantitative data were expressed as mean±SEM. Statistical analysis was performed with the Student t test. Differences were considered significant when probability values were <0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Conditional Induction of c-Jun in HUVECs
To study the functional role of c-Jun in EC apoptosis, tetracycline-regulated adenoviral expression vectors were generated, in which the full-length coding region of human c-jun cDNA was cloned into the E1- and E3-deleted adenovirus 5. Expression of the c-jun gene, driven by a 7x tet/minimal cytomegalovirus promoter, was under the control of an artificial tTA, which was produced by a helper adenovirus, AdtTA. The principle of this regulatable system is that an appropriate level of tetracycline can disrupt interaction between the transactivator and the tTA-responsive element and, therefore, shut off the induced gene expression.⁷ As shown in Figure 1, c-jun expression was induced at both mRNA and protein levels in HUVECs coinfected with AdJun and AdtTA. Expression of c-jun transcripts can be detected as early as 12 hours after the transfection and remains overexpressed for the remainder of the experiments. Addition of tetracycline into the culture medium tightly controlled the induced c-jun expression at both mRNA and protein levels in a dose-dependent manner. In contrast, the abundance of VWF mRNA and JNK-1 protein was not affected by tetracycline-regulated c-Jun expression. Tetracycline, at tested concentrations (0.1 µg/mL), did not show either cytotoxicity (eg, cell morphology, viability, and growth) or interference with expression of endogenous genes thus far tested in HUVECs.

View larger version (64K): [in this window] [in a new window]   Figure 1. Regulated expression of transcription factor c-Jun in human ECs. HUVECs were coinfected with AdtTA and AdJun or Adß-gal as control and incubated for 24 hours in the presence of tetracycline (Tc, 0–0.1 µg/mL). A, Northern blot was hybridized to cDNA probes for human c-jun and VWF genes. B, Western blot was reacted with antibodies against human c-Jun or JNK-1.

c-Jun Triggers Apoptosis in HUVECs
Using conditional c-jun expression in HUVECs, we examined the role of c-Jun in EC apoptosis. Confluent cells were infected with AdJun and AdtTA and incubated in medium containing 20% FBS in the presence or absence of tetracycline (0.1 µg/mL). Alternatively, cells were coinfected with Adß-gal and AdtTA as a control for adenovirus-mediated gene overexpression. Starting at 24 hours after the infection, the first morphological changes, including membrane blebbing, nuclear condensation, and cell detachment, were observed in HUVECs overexpressing c-Jun (Figure 2A). The number of apoptotic cells increased with time and accounted for 50% of the total cell population by 48 hours after infection. In contrast, such apoptotic morphological changes did not occur in ECs overexpressing the ß-galactosidase gene or those identically infected with Ad-Jun but maintained in tetracycline-supplemented medium.

View larger version (56K): [in this window] [in a new window]   Figure 2. Endothelial apoptosis induced by c-Jun. HUVECs were coinfected with AdtTA and AdJun in the presence or absence of Tc (0.1 µg/mL). Alternatively, HUVECs were infected with AdtTA and Adßgal as a vector control. Forty-eight hours after the infection, cells were subjected to different assays for apoptosis. Apoptotic characteristics were exclusively detected in HUVECs overexpressing the c-jun gene. A, Light microscopy showed cell shrinkage and nuclear condensation. B, TUNEL assay identifies apoptotic cells manifested as green fluorescein (b, Adß-gal; c, AdJun; and d, AdJun+Tc). Nuclei were counterstained with propidium iodide (orange). Overexpression of ß-gal was detected with in situ X-gal staining (a). C, Internucleosomal DNA cleavage on c-Jun overexpression. DNA was separated by 1.5% agarose gel, stained with ethidium bromide, and compared with DNA standard (Marker, 100-bp ladder). A characteristic ladder pattern is present in ECs overexpressing c-Jun. D, Cytosolic lysates were quantitatively determined for DNA fragmentation with an ELISA specific for histone-associated DNA fragments (mean±SEM). *P<0.05 compared with control. Data are expressed as fold increase of control. Tc indicates tetracycline.

A series of assays was performed to document this c-Jun–triggered EC death as an apoptotic process. These include the TUNEL assay (Figure 2B), DNA laddering (Figure 2C), and an ELISA specific for histone-associated DNA fragments (Figure 2D). These typical apoptosis determinations occurred exclusively in ECs overexpressing c-jun. Collectively, our results point directly to a causative role for c-jun in EC apoptosis.

Caspase Activity Is Involved in c-Jun–Triggered EC Apoptosis
The caspase family of cysteine proteases is increasingly implicated in the apoptotic process for numerous cell types, including ECs. To examine whether c-Jun–triggered apoptosis involves activation of caspase, protein isolated from ECs undergoing c-Jun–triggered apoptosis was subjected to Western blot analysis using an antibody raised against PARP. As shown in Figure 3A, marked PARP cleavage was identified in cells overexpressing c-Jun but not in controls. This finding suggests the involvement of caspase activation. Furthermore, we tested whether c-Jun–induced EC apoptosis could be inhibited by specific peptide inhibitors of caspases. As shown in Figure 3B, c-Jun–induced EC apoptosis was largely attenuated by zDEVD.fmk, an irreversible cell-permeable inhibitor of caspase-3. Under the same conditions, the addition of zVAD.fmk, a relatively specific inhibitor of caspase-1, showed less inhibitory effect. Thus, it is highly suggested that activation of caspases, especially caspase-3, may mediate c-Jun–induced EC apoptosis.

View larger version (28K): [in this window] [in a new window]   Figure 3. Caspase activation. A, PARP was detected by Western analysis with a rabbit antiserum against PARP. The cleaved fragments of PARP were detected in HUVECs overexpressing c-jun. B, Effects of caspase inhibitors on c-Jun–induced apoptosis. After infection with AdJun, HUVECs were seeded in 96-well plates and incubated for 24 hours in the presence of vehicle or caspase inhibitors (10 µmol/L). Cell survival was assessed by the MTT assay, and viability was expressed relative to noninfected cells. The values shown are mean±SEM of triplicate samples. *P<0.05 vs the cells overexpressing c-Jun in the absence of caspase inhibitor or vehicle.

The N-Terminal Transactivation Domain Is Required for the Proapoptotic Effect of c-Jun
As an important member of the transcription factor activator protein-1 (AP-1), c-Jun, in the form of heterodimers or homodimers, can transcriptionally regulate gene expression via its amino-terminal transactivation domain.¹³ To test whether the proapoptotic effect of c-Jun was associated with its transactivation ability, an adenovirus expressing TAM67, a mutant form of c-Jun lacking the transactivation domain, was used to infect ECs. The truncated c-jun gene was overexpressed in HUVECs. In contrast to wild-type c-Jun, TAM67 did not cause apoptosis in HUVECs (Figure 4). Thus, c-Jun may function as a proapoptotic molecule, likely via its transcriptional control of target genes essential to cell death.

View larger version (18K): [in this window] [in a new window]   Figure 4. Cell death induced by wild-type and mutant c-jun. A, Cell viability was assessed by the MTT assay at 48 hours after infection with either AdJun (wild-type) or AdTAM67 (c-Jun 3-122) at increasing MOI. Shown is the relative viability expressed as the mean±SEM of triplicate samples. B, Northern blot shows overexpression of truncated c-jun gene in HUVECs. Total RNA was isolated from control cells or cells infected with AdTAM67 or control vector. Results were confirmed by 3 separate experiments.

c-Jun Is Involved in H₂O₂-Induced EC Apoptosis
Clinical studies as well as experimental evidence suggest a causal pathophysiological role of increased oxidative stress in endothelial dysfunction and injury. H₂O₂ can be produced extracellularly via the respiratory burst of neutrophils or macrophages or intracellularly by the activation of the xanthine oxidase system and has been demonstrated to be a proapoptotic stimulus for ECs.¹⁴ In this study, we explored the involvement of c-Jun/AP-1 activation in H₂O₂-induced EC apoptosis. Incubation with H₂O₂ (200 µmol/L) for 18 hours induced apoptosis of ECs, as previously reported. DAPI staining revealed nuclear condensation and fragmentation typical of apoptosis (Figure 5B). Western blot analysis demonstrated that upregulation of c-Jun protein was induced in response to H₂O₂ treatment (Figure 5A). To examine whether activation of c-jun is required for H₂O₂-induced EC apoptosis, HUVECs were infected with AdTAM67, which selectively inactivated c-Jun/AP-1 via a trans-dominant-negative mechanism, and exposed to H₂O₂ for 18 hours. The susceptibility of TAM67-expressing cells to H₂O₂-induced apoptosis was assessed and compared with ECs infected with the adenovirus expressing a wild-type c-jun or that infected with Ad-gal. As shown in Figure 5C, TAM67-expressing ECs exhibited resistance to H₂O₂-induced apoptosis, whereas ECs overexpressing wild-type c-Jun showed increased susceptibility to H₂O₂-induced apoptosis. Therefore, it is suggested that the proapoptotic signaling of H₂O₂ may be mediated through an endogenous c-jun/AP-1 pathway.

View larger version (23K): [in this window] [in a new window]   Figure 5. Involvement of c-Jun activation in H2O2-induced EC apoptosis. A, Expression of c-Jun in response to H2O2 exposure. HUVECs were exposed to 200 µmol/L H2O2 for indicated periods, and the expression of c-Jun was detected by Western blot analysis. B, Apoptosis was detected by fluorescence staining of nuclei after exposure to H2O2 for 18 hours (arrowheads indicate apoptotic cells). C, Effect of inactivation of c-Jun on H2O2-induced EC apoptosis. Twenty-four hours after infection with Adß-gal, AdTAM67, or AdJun (100 MOI), cells were treated with (hatched bars) or without (filled bars) H2O2 for 18 hours and subjected to the MTT assay. H2O2-induced apoptosis was expressed as viability relative to nontreated cells. Values are mean±SEM obtained from 3 separate experiments. *P<0.05 vs control (infected with Adß-gal and treated with H2O2).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we have used a regulated adenoviral expression system to evaluate the functional role of c-Jun in HUVECs. Using this approach, we provide the first direct evidence that c-jun can be a potent apoptosis inducer in primary cultured human endothelial cells.

Because ECs, especially human primary ECs, are refractory to many transfection techniques,^{15 16} adenoviral vectors have been used to confer high levels of transgene expression.^{17 18} Nevertheless, caution must be used when interpreting the results obtained with such an approach, especially those regarding cell death. For example, in addition to the adenoviral early protein E1,¹⁹ some late proteins have recently been shown to induce apoptosis.^{20 21} Thus, it is critical to determine the possibility that apoptosis may be caused by the adenoviral vector itself rather than by the effect of transgene expression. In this study, we have examined this possibility in 2 ways. First, AdJun–mediated EC apoptosis could be completely abolished in identically infected cells by stopping the c-jun expression. Second, high levels of ß-galactosidase expression did not induce apoptosis. Thus, EC apoptosis has been demonstrated to be a consequence of specific c-Jun induction.

c-Jun is a major component of the AP-1 transcriptional complex. It can form either homodimers or heterodimers with other AP-1 components from the Jun family (Jun B and Jun D) or the Fos family (c-Fos, Fra-1, Fra-2, and Fos B), and it binds to a palindromic sequence known as the tissue-type plasminogen activator–responsive element or AP-1 consensus site.²² In addition, c-Jun can also dimerize with members of the activating transcription factor family and can bind to a sequence recognized by the cAMP-responsive element binding protein family.²³ Thus, the different c-Jun dimers can exhibit distinct transcriptional properties with diverse biological consequences. Evidence has accumulated indicating a role for c-Jun/AP-1 in apoptosis, functioning as both a positive and a negative modulator of apoptotic pathways in different cell types.^{23 24} For example, when rat sympathetic neurons undergo apoptosis during nerve growth factor withdrawal, the levels of c-jun mRNA and protein increase.^{25 26} The microinjection of neutralizing antibodies specific for c-Jun, or overexpression of a dominant-negative c-jun mutant, was able to protect nerve growth factor–deprived sympathetic neurons from apoptosis. However, somewhat contradictory evidence has been published. c-Jun has been suggested not to be essential for apoptosis in vivo during normal development, as c-jun^–/– mouse embryos exhibited increased rather than reduced apoptosis in their livers. These authors conclude that AP-1 may have a protective role against apoptosis.²⁷ Several studies in other cell types also showed that c-Jun induction either has no direct relationship to apoptosis or has an inhibitory role.^{24 28} Recently, Bossy-Wetzel et al²⁹ provided more direct observations. Conditional induction of c-Jun activity appears sufficient to trigger apoptotic cell death in the NIH 3T3 murine fibroblast cell line. However, similarly generated BALB/c 3T3 cell lines expressed high levels of c-Jun but did not undergo apoptosis,²⁹ which underscores the necessity of considering the genetic background and cellular context when evaluating the role of c-Jun in apoptosis. Therefore, we present not only the first direct evidence implicating c-Jun as a proapoptotic molecule in vascular endothelial cells, but also the first report extending the finding from a rodent cell line to human primary cells. Hence, this observation may have unique and important implications for human vascular biology.

Increasing evidence has emerged linking EC apoptosis to various vascular pathologic conditions, including atherogenic and thrombotic processes. Apoptotic ECs have been detected at the luminal surface of atherosclerotic coronary vessels. Circulating ECs, suggesting EC death, were detected in hypertension³⁰ and homocysteinemia.³¹ Additionally, the molecular markers of apoptosis, such as Fas, were found on ECs in transplant atherosclerosis. In vitro studies show cultured ECs undergoing apoptosis on exposure to various stimuli, which are shown to promote atherogenesis or its complications. EC proapoptotic factors include proinflammatory cytokines, lipopolysaccharides, LDL oxidative products, cholesterol metabolites, homocysteine,³² high levels of proinsulin and glucose,³³ angiotensin II,³⁴ activated leukocytes,³⁵ and oxidative stress.⁴ Furthermore, it has been demonstrated that apoptotic vascular ECs become procoagulant³⁶ and hyperadhesive for monocytic cells.³⁷ It is indicated that EC apoptosis, as a pathological mechanism, may convert ECs to a prothrombotic and proinflammatory state. This in turn may contribute to the pathogeneses of important vascular processes, including angiogenesis, inflammation, thrombosis, and atherosclerosis.

Recent studies have suggested that H₂O₂, a reactive compound formed endogenously in the breakdown of superoxide or generated by inflammatory cells, may mediate the induction of apoptosis in various cell types, including ECs.³⁸ Despite its importance in the physiopathology of the vascular endothelium, the molecular mechanisms regulating the H₂O₂ injury of ECs are currently not well understood. The results obtained from other types of cells indirectly lead to speculation that c-Jun activation may be involved in H₂O₂-induced EC apoptosis. First, c-Jun/AP-1 has been known as a major transcription factor responsive to the cellular redox state. Second, both c-Jun expression and AP-1 binding activity can be induced by H₂O₂. In addition, in glomerular mesangial cells, disruption of c-Jun/AP-1 inhibited H₂O₂-initiated apoptosis.³⁹ In this study, immunoblot analysis revealed an up-regulation of c-Jun in HUVECs after H₂O₂ treatment. This is consistent with a previous observation in human microvascular ECs that H₂O₂ induced an AP-1 binding complex containing c-Jun.⁴⁰ A functional role of c-Jun/AP-1 activation in H₂O₂-induced apoptosis has been further established by using adenovirus-mediated expression of c-jun or its dominant-negative mutant, TAM67. When c-Jun is preinduced, ECs appear to be predisposed to H₂O₂ damage, whereas disrupting c-Jun activity by TAM67 renders ECs resistant to the H₂O₂ proapoptotic effect. Together, these data strongly indicate a pathophysiological role for c-Jun in EC apoptosis triggered by oxidative stress. On the other hand, as shown in Figure 5C, the H₂O₂ proapoptotic effect was largely but not completely abolished in TAM67-overexpressing cells. It is likely that an alternative pathway, other than c-Jun/AP-1, may also mediate the H₂O₂ damage to ECs.

How might c-Jun trigger EC apoptosis? First, overexpression of c-Jun leads to specific cleavage of PARP (Figure 3), which is a nuclear protein and a major substrate for caspase-3.⁴¹ Its characteristic cleavage is an indicator of caspase-3 activation. Moreover, the proapoptotic effect of c-Jun can be effectively attenuated by peptide inhibitors of caspases, particularly zDEVD.fmk, which is a selective inhibitor of caspase-3. Thus, a caspase cascade may be responsible for the c-Jun–triggered apoptotic program. However, the downstream mechanisms by which c-Jun initiates the caspase cascade remain to be identified. c-Jun is a transcriptional regulator of gene expression. Hypothetically, c-Jun may trigger EC apoptosis by activating a variety of "death genes," including members of the caspase family. Alternatively, c-Jun may repress some "protective genes." Such regulatory mechanisms may be achieved either directly, via the AP-1 cis elements in target genes, or indirectly, via the interaction with other transcription factors. The fact that deletion of the transactivation domain of c-Jun abolished this proapoptotic effect (Figure 4) supports such a hypothesis. Interestingly, a recent report described overexpression in rat arterial endothelium of transforming growth factor-ß, an AP-1–responsive gene, caused massive apoptosis.⁴² It is thus possible that some of the c-jun/AP-1–dependent genes may participate in promoting EC apoptosis. Apparently, future studies identifying c-Jun–responsive death genes will be imperative to elucidate the downstream mechanisms of c-Jun–triggered EC apoptosis.

In conclusion, we have demonstrated that, in human ECs, c-jun is a proapoptotic gene and the proapoptotic effect of c-Jun involves its capability for transcriptional regulation and activation of the caspases. Furthermore, c-Jun/AP-1 activation is an important mediator in EC apoptosis induced by oxidative stress, such as H₂O₂. Thus, our current data may provide insight into understanding the molecular mechanisms underlying vascular endothelial apoptosis and designing future therapeutic intervention of pathological conditions involving vascular endothelial injury.

	Acknowledgments

 
This work was supported by National Institutes of Health Grant HL 43023 (to M.B.S.).

Received December 21, 1998; accepted June 16, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Karsan A, Harlan JM. Modulation of endothelial cell apoptosis: mechanisms and pathophysiological roles. J Atheroscler Thromb. 1996;3:75–80.[Medline] [Order article via Infotrieve]

2. Karsan A, Yee E, Harlan JM. Endothelial cell death induced by tumor necrosis factor- is inhibited by the Bcl-2 family member, A1. J Biol Chem. 1996;271:27201–27204.[Abstract/Free Full Text]

3. Choi KB, Wong F, Harlan JM, Chaudhary PM, Hood L, Karsan A. Lipopolysaccharide mediates endothelial apoptosis by a FADD-dependent pathway. J Biol Chem. 1998;273:20185–20188.[Abstract/Free Full Text]

4. Suhara T, Fukuo K, Sugimoto T, Morimoto S, Nakahashi T, Hata S, Shimizu M, Ogihara T. Hydrogen peroxide induces up-regulation of Fas in human endothelial cells. J Immunol. 1998;160:4042–4047.[Abstract/Free Full Text]

5. Harada-Shiba M, Kinoshita M, Kamido H, Shimokado K. Oxidized low density lipoprotein induces apoptosis in cultured human umbilical vein endothelial cells by common and unique mechanisms. J Biol Chem. 1998;273:9681–9687.[Abstract/Free Full Text]

6. Rauscher FJ, Cohen DR, Curran T, Bos TJ, Vogt PK, Bohmann D, Tjian R, Franza BRJ. Fos-associated protein p39 is the product of the jun proto-oncogene. Science. 1988;240:1010–1016.[Abstract/Free Full Text]

7. Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A. 1992;89:5547–5551.[Abstract/Free Full Text]

8. 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]

9. 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]

10. Auer KL, Contessa J, Brenz-Verca S, Pirola L, Rusconi S, Cooper G, Abo A, Wymann MP, Davis RJ, Birrer M, Dent P. The Ras/Rac1/Cdc42/SEK/JNK/c-Jun cascade is a key pathway by which agonists stimulate DNA synthesis in primary cultures of rat hepatocytes. Mol Biol Cell. 1998;9:561–573.[Abstract/Free Full Text]

11. Graham FL, Prevec L. Adenovirus-based expression vectors and recombinant vaccines. Biotechnology. 1992;20:363–390.[Medline] [Order article via Infotrieve]

12. Zhu Y, Lin JH, Liao HL, Friedli OJ, 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]

13. Bohmann D, Tjian R. Biochemical analysis of transcriptional activation by Jun: differential activity of c- and v-Jun. Cell. 1989;59:709–717.[Medline] [Order article via Infotrieve]

14. Harrison DG. Endothelial function and oxidant stress. Clin Cardiol. 1997;20:II-7.

15. Sawai H, Okazaki T, Yamamoto H, Okano H, Takeda Y, Tashima M, Sawada H, Okuma M, Ishikura H, Umehara H. Requirement of AP-1 for ceramide-induced apoptosis in human leukemia HL-60 cells. J Biol Chem. 1995;270:27326–27331.

16. Tanner FC, Carr DP, Nabel GJ, Nabel EG. Transfection of human endothelial cells. Cardiovasc Res. 1997;35:522–528.[Abstract/Free Full Text]

17. Merrick AF, Shewring LD, Sawyer GJ, Gustafsson KT, Fabre JW. Comparison of adenovirus gene transfer to vascular endothelial cells in cell culture, organ culture, and in vivo. Transplantation. 1996;62:1085–1089.[Medline] [Order article via Infotrieve]

18. Wrighton CJ, Hofer-Warbinek R, Moll T, Eytner R, Bach FH, de Martin R. Inhibition of endothelial cell activation by adenovirus-mediated expression of IB, an inhibitor of the transcription factor NF-B. J Exp Med. 1996;183:1013–1022.[Abstract/Free Full Text]

19. Nakajima T, Yageta M, Shiotsu K, Morita K, Suzuki M, Tomooka Y, Oda K. Suppression of adenovirus E1A-induced apoptosis by mutated p53 is overcome by coexpression with Id proteins. Proc Natl Acad Sci U S A. 1998;95:10590–10595.[Abstract/Free Full Text]

20. Lavoie JN, Nguyen M, Marcellus RC, Branton PE, Shore GC. E4orf4, a novel adenovirus death factor that induces p53-independent apoptosis by a pathway that is not inhibited by zVAD-fmk. J Cell Biol. 1998;140:637–645.[Abstract/Free Full Text]

21. Agah R, Kirshenbaum LA, Abdellatif M, Truong LD, Chakraborty S, Michael LH, Schneider MD. Adenoviral delivery of E2F-1 directs cell cycle reentry and p53-independent apoptosis in postmitotic adult myocardium in vivo. J Clin Invest. 1997;100:2722–2728.[Medline] [Order article via Infotrieve]

22. Angel P, Karin M. The role of Jun, Fos and the AP-1 complex in cell proliferation and transformation. Biochim Biophys Acta. 1991;1072:129–157.[Medline] [Order article via Infotrieve]

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. Liebermann DA, Gregory B, Hoffman B. AP-1 (Fos/Jun) transcription factors in hematopoietic differentiation and apoptosis. Int J Oncol. 1998;12:685–700.[Medline] [Order article via Infotrieve]

25. Estus S, Zaks WJ, Freeman RS, Gruda M, Bravo R, Johnson EMJ. Altered gene expression in neurons during programmed cell death: identification of c-jun as necessary for neuronal apoptosis. J Cell Biol. 1994;127:1717–1727.[Abstract/Free Full Text]

26. Ham J, Babij C, Whitfield J, Pfarr CM, Lallemand D, Yaniv M, Rubin LL. A c-Jun dominant negative mutant protects sympathetic neurons against programmed cell death. Neuron. 1995;14:927–939.[Medline] [Order article via Infotrieve]

27. Roffler-Tarlov S, Brown JJ, Tarlov E, Stolarov J, Chapman DL, Alexiou M, Papaioannou VE. Programmed cell death in the absence of c-Fos and c-Jun. Development. 1996;122:1–9.[Abstract]

28. Shimizu R, Komatsu N, Nakamura Y, Nakauchi H, Nakabeppu Y, Miura Y. Role of c-jun in the inhibition of erythropoietin receptor-mediated apoptosis. Biochem Biophys Res Commun. 1996;222:1–6.[Medline] [Order article via Infotrieve]

29. Bossy-Wetzel E, Bakiri L, Yaniv M. Induction of apoptosis by the transcription factor c-Jun. EMBO J. 1997;16:1695–1709.[Medline] [Order article via Infotrieve]

30. Gobe G, Browning J, Howard T, Hogg N, Winterford C, Cross R. Apoptosis occurs in endothelial cells during hypertension-induced microvascular rarefaction. J Struct Biol. 1997;118:63–72.[Medline] [Order article via Infotrieve]

31. Hladovec J, Sommerova Z, Pisarikova A. Homocysteinemia and endothelial damage after methionine load. Thromb Res. 1997;88:361–364.[Medline] [Order article via Infotrieve]

32. Rounds S, Yee WL, Dawicki DD, Harrington E, Parks N, Cutaia MV. Mechanism of extracellular ATP- and adenosine-induced apoptosis of cultured pulmonary artery endothelial cells. Am J Physiol. 1998;275:L379–L388.[Abstract/Free Full Text]

33. Du XL, Sui GZ, Stockklauser-Farber K, Weiss J, Zink S, Schwippert B, Wu QX, Tschope D, Rosen P. Introduction of apoptosis by high proinsulin and glucose in cultured human umbilical vein endothelial cells is mediated by reactive oxygen species. Diabetologia. 1998;41:249–256.[Medline] [Order article via Infotrieve]

34. Dimmeler S, Rippmann V, Weiland U, Haendeler J, Zeiher AM. Angiotensin II induces apoptosis of human endothelial cells. Protective effect of nitric oxide. Circ Res. 1997;81:970–976.[Abstract/Free Full Text]

35. Lindner H, Holler E, Ertl B, Multhoff G, Schreglmann M, Klauke I, Schultz-Hector S, Eissner G. Peripheral blood mononuclear cells induce programmed cell death in human endothelial cells and may prevent repair: role of cytokines. Blood. 1997;89:1931–1938.[Abstract/Free Full Text]

36. Bombeli T, Karsan A, Tait JF, Harlan JM. Apoptotic vascular endothelial cells become procoagulant. Blood. 1997;89:2429–2442.[Abstract/Free Full Text]

37. Hebert MJ, Gullans SR, Mackenzie HS, Brady HR. Apoptosis of endothelial cells is associated with paracrine induction of adhesion molecules: evidence for an interleukin-1ß-dependent paracrine loop. Am J Pathol. 1998;152:523–532.[Abstract]

38. Hermann C, Zeiher AM, Dimmeler S. Shear stress inhibits H₂O₂-induced apoptosis of human endothelial cells by modulation of the glutathione redox cycle and nitric oxide synthase. Arterioscler Thromb Vasc Biol. 1997;17:3588–3592.[Abstract/Free Full Text]

39. Ishikawa Y, Yokoo T, Kitamura M. c-Jun/AP-1, but not NF-B, is a mediator for oxidant-initiated apoptosis in glomerular mesangial cells. Biochem Biophys Res Commun. 1997;240:496–501.[Medline] [Order article via Infotrieve]

40. Shono T, Ono M, Izumi H, Jimi SI, Matsushima K, Okamoto T, Kohno K, Kuwano M. Involvement of the transcription factor NF-B in tubular morphogenesis of human microvascular endothelial cells by oxidative stress. Mol Cell Biol. 1996;16:4231–4239.[Abstract]

41. Duriez PJ, Shah GM. Cleavage of poly(ADP-ribose) polymerase: a sensitive parameter to study cell death. Biochem Cell Biol. 1997;75:337–349.[Medline] [Order article via Infotrieve]

42. Schulick AH, Taylor AJ, Zuo W, Qiu C, Dong G, Woodward RN, Agah R, Roberts AB, Virmani R, Dichek DA. Overexpression of transforming growth factor ß₁ in arterial endothelium causes hyperplasia, apoptosis, and cartilaginous metaplasia. Proc Natl Acad Sci U S A. 1998;95:6983–6988.[Abstract/Free Full Text]

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