Free Radical Biology & Medicine, Vol. 37, No. 4, pp. 539 –548, 2004Copyright D 2004 Elsevier Inc.

Printed in the USA. All rights reserved0891-5849/$-see front matter

doi:10.1016/j.freeradbiomed.2004.05.011

Original Contribution

INDUCTION OF ENDOTHELIAL iNOS BY 4-HYDROXYHEXENAL

THROUGH NF-nB ACTIVATION

J. Y. LEE,* J. H. JE,y K. J. JUNG,y B. P. YU,z and H. Y. CHUNG*,y

*Genetic Engineering Research Institute and yCollege of Pharmacy, Aging Tissue Bank, Pusan National University,Busan 609-735, South Korea; and zDepartment of Physiology,

The University of Texas Health Science Center, San Antonio, TX, USA

(Received 29 January 2004; Revised 22 April 2004; Accepted 14 May 2004)

Available online 5 June 2004

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Abstract—Lipid peroxidation and its end-product, 4-hydroxyhexenal (HHE), are known to affect redox balance during

aging, which causes various degenerative processes including vascular alterations from endothelial cell deterioration. To

better understand the molecular action of HHE in the development of vascular abnormalities during the aging process, we

investigated whether the upregulation of inducible endothelial nitric oxide synthase (iNOS) by HHE is mediated through

nuclear factor nB (NF-nB) activation. Results indicate that HHE stimulates iNOS by the transcriptional regulation of NF-

nB activation through cytosolic nB degradation inhibitors (InB). Pretreatment with NF-nB inhibitors Bay 11-7082 and

N-acetyl cysteine (NAC) suppressed the upregulation of iNOS by blunting InB degradation and NF-nB binding activity.

Because inflammatory stimuli induce iNOS to generate large amounts of nitric oxide (NO), intracellular NO levels in the

presence of Bay 11-7082, NAC, and caffeic acid methyl ester were estimated. These inhibitors significantly suppressed

the HHE-induced NO levels to a basal level. These findings strongly suggest that in endothelial cells, HHE induces iNOS

gene expression through NF-nB activation, which can lead to vascular dysfunction by the activation of various

proinflammatory genes. D 2004 Elsevier Inc. All rights reserved.

Keywords—4-Hydroxyhexenal, iNOS, NF-nB, Endothelial cells, NO, Vascular dysfunction, Aging, Free radicals

INTRODUCTION

Lipid molecules are particularly vulnerable to oxidative

attack because their unstable reactive double bonds can

set off a series of peroxidative chain reactions [1]. Lipid

peroxidation is known to produce many reactive species

(RS), such as 4-hydroxynonenal (HNE), which is known

to inflict cell damage, redox disturbance [1–3], and

various other deleterious processes [4–8]. Included in

these processes is HNE’s ability to disrupt vascular Ca2+

homeostasis [9] and inflict injury on vascular smooth

muscle [10].

ress correspondence to: Hae Young Chung, Department of

cy, College of Pharmacy, Aging Tissue Bank, Pusan National

sity, Jangjun-dong, Gumjung-ku, Busan 609-735, South Korea;

2-51-518-2821; E-mail: hyjung@pusan.ac.kr.

539

Moreover, by virtue of their high reactivity, HNE and

other reactive aldehydes modify the regulation of cell

signaling and the induction of oxidative stress-mediated

apoptosis [11–13]. In vascular smooth muscle cells,

HNE is reported to activate nuclear factor nB (NF-nB)and thereby promote apoptotic cell death [14]. However,

at present, the mechanisms underlying endothelial cell

dysfunction through the regulation of NF-nB activation

by another reactive aldehyde, 4-hydroxyhexenal (HHE),

are not delineated.

HHE is a reactive byproduct of n-3 fatty acid

peroxidation [1,15] and is structurally similar to

HNE, which is derived from n-6 fatty acids, but its

biological actions and efficacies may vary greatly from

HHE. For instance, Kristal et al. found that compared

to HNE, HHE is extremely more effective in inducing

mitochondrial permeability transition (MPT), which

leads to disrupted calcium homeostasis [16]. HHE also

was demonstrated to be highly effective in its ability to

J. Y. Lee et al.540

inhibit the mitochondria ATP translocator [17]. In view of

recent reports [18,19] on oxidant-induced mitochondrial

dysfunction and increased MPT activation, reactive alde-

hydes like HHE are expected to have a strong impact on

mitochondrial depolarization in human aortic endothelial

apoptosis.

Yamada et al. reported endogenous production of

HHE in human atherosclerotic lesions [20]. Although

the potential harmful actions of HHE were predicted, at

present, little information is available on the molecular

mechanisms by which HHE interacts with the vascular

endothelial system. In this regard, it is worth mentioning

our most recent work on the effect of HHE induction in

vascular endothelial apoptosis [21].

Inducible nitric oxide synthase (iNOS) is regulated at

the transcriptional level, and its gene promoter has

binding sites for multiple transcription factors, including

NF-nB [22]. Although iNOS has been implicated in

altered vascular activities, such as in inflammation [23]

and diabetes [24], the effects of HHE on the molecular

mechanisms of iNOS gene expression that lead to nitric

oxide (NO) generation in endothelial cells have not been

explored.

The redox-sensitive transcription factor NF-nBplays an important role in the expression of a variety

of genes involved in inflammatory responses and

apoptosis in multiple tissues and cell types [25,26].

For example, NF-nB activation has been implicated in

gene modulation of the inflammatory responses iNOS

and cyclooxygenase-2 (COX-2) [27,28] and in vascu-

lar diseases [29].

The present study was launched to seek molecular

information about vascular dysfunction caused by

HHE by way of NF-nB activation. We attempted to

determine whether the upregulation of iNOS by HHE

in endothelial cells is mediated by NF-nB activation.

MATERIALS AND METHODS

Culture conditions and HHE treatments

YPEN-1, rat prostatic endothelial cells, was

obtained from ATCC (American Type Culture Collec-

tion, Manassas, VA, USA). The cells were grown in

Dulbecco’s modified Eagle medium (Nissui, Tokyo,

Japan) containing 2 mM L-glutamine, 100 mg/ml

streptomycin, 2.5 mg/l amphotericin B, and 5% heat-

inactivated fetal bovine serum. Cells were maintained

at 37jC in a humidified atmosphere containing 5%

CO2/95% air. Cells were discarded after 3 months at

which time new cells were obtained from frozen stock.

Cells at exponential phase were used for all experi-

ments, and cell viability (>90%) was assessed by

trypan blue exclusion.

A commercial HHE (purity >98%; Cat. No. 32060)

was obtained from Cayman Chemical, Inc. Working

solutions of HHE (the final concentration never exceed-

ing the 0.1% level of Ethanol) were made in phosphate-

buffered saline (PBS) immediately before use. For all

experiments, cells were plated in 100 mm culture dishes,

and cultures at 70–80% confluence were used for the

chemical exposures. After a 24 h attachment period,

media were replaced with serum-free media, and cells

were treated with 30 AM HHE after preincubation for 30

min with various inhibitors. After 1 to 24 h periods, cells

were harvested with ice-cold PBS. Cell lysates were used

for Western blot analysis.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide (MTT) assay for cell viability

Cell survival was quantified by colorimetric MTT

(Sigma, St. Louis, MO, USA) assay, which measures

mitochondrial activity in viable cells. This method is

based on the conversion of the MTT to MTT-formazan

crystal by mitochondrial enzyme.

Preparation of cytosolic and nuclear extracts

Nuclear and cytosolic extracts were prepared accord-

ing to Deng et al. [30]. Treated cells were washed and

then scraped into 1.0 ml of ice-cold PBS and pelleted at

3000 rpm for 5 min at 4jC. The pellets were suspended

in 10 mM Tris (pH 8.0), with 1.5 mM MgCl2, 1 mM

dithiothreitol (DTT), 0.1% Nonidet P-40 (NP-40), and

inhibitors, and incubated on ice for 15 min. Nuclei were

separated from cytosol by centrifugation at 12,000 rpm

for 15 min at 4jC. The supernatants (cytosolic fraction)

were removed, and the pellets were suspended in 10 mM

Tris (pH 8.0) with 50 mM KCl, 100 mM NaCl, and

inhibitors, incubated on ice for 30 min, and then centri-

fuged at 12,000 rpm for 30 min at 4jC (nuclear

fraction).

Measurements of transfection and luciferase reporter

assay for NF-jB activity

NF-nB activity was examined using a luciferase

plasmid DNA, pTAL-NF-nB, that contains a specific

binding sequence for NF-nB (BD Biosciences Clontech,

Palo Alto, CA, USA) [31]. Transfection was carried out

using FuGENE 6 Reagent (Roche, Indianapolis, IN,

USA). Briefly, 5 � 104 cells per well were seeded in

24 well plates and transfected with 0.2 Ag DNA/0.5

Al FuGENE 6 complexes. After transfection, cells were

treated with HHE or malondialdehyde (MDA) after being

preincubated for 20 min with Bay 11-7082, N-acetylcys-

teine (NAC), or caffeic acid methyl ester (CAPE) in

serum-free medium. After additional incubation for 6 h,

cells were washed with PBS and added to the plate using

the Steady-Glo Luciferase Assay System (Promega,

Fig. 1. Effects of HHE on endothelial cell viability. Cells wereincubated in serum-free medium at HHE concentrations indicated inorder to avoid the HHE binding affinity of albumin. After 1–24 h ofexposure, cell viability was assessed by MTT assay as described underMaterials and Methods. The results are presented as means F SE ofthree individual experiments. Statistical significance: *p < .05 vs.control.

Induction of iNOS by HNE via NF-nB 541

Madison, WI, USA). Luciferase activity was measured

by a luminometer (GENious; Tecan, Salzburg, Austria).

The obtained raw luciferase activities were normalized

by protein concentration per each well.

Analysis of proteins by Western blot

Western blotting was carried out as described previ-

ously [32]. The cells were harvested, washed twice with

ice-cold PBS, and lysed in a TNN buffer (50 mM Tris–

HCl, pH 8.0, 120 mM sodium chloride, 0.5% NP-40) that

was supplemented with protease inhibitors (2 Ag/ml

aprotinin, 2 Ag/ml leupeptin, 100 Ag/ml PMSF, 5 Ag/ml

pepstatin, and 1 mM DTT) and phosphate inhibitors (20

mM NaF and 2 mM Na3VO4) for 1 h on ice, vortexing

after every 10 min. Lysates were centrifuged at 12,000

rpm for 30 min to remove insoluble material. The protein

concentration was determined by the Lowry method

(Sigma) using BSA as a standard. Equal amounts of

protein were separated on 6–15% SDS–PAGE gels. The

gels were subsequently transferred onto a nitrocellulose

membrane (Hybond C; Amersham Corp.). Polyclonal

antibodies to NF-nB (p65), InBa, and iNOS were pur-

chased from Santa Cruz Biotechnology, Inc. (Santa Cruz,

CA, USA). Monoclonal sheep anti-mouse IgG or donkey

anti-rabbit IgG horseradish peroxidase-conjugated sec-

ondary antibodies were used at 1:1000. Proteins were

detected by an enhanced chemiluminescence reagent

using a commercial kit (Amersham Life Science).

Electrophoretic mobility shift assay

The electrophoretic mobility shift assay (EMSA) was

used to characterize the binding activities of NF-nB in

nuclear extracts [33]. Protein–DNA binding mixture

containing 20 Ag of nuclear protein extract was incubat-

ed for 20 min at 4jC in binding medium containing 5%

glycerol, 1 mM MgCl2, 50 mM NaCl, 0.5 mM EDTA, 2

mM DTT, 1% NP-40, 10 mM Tris (pH 7.5), and 1 Ag of

poly(dI-dC)�poly(dI-dC). Radiolabeled transcription fac-

tor consensus oligonucleotide (20,000 cpm of 32P) was

added, and the complete mixture was incubated for an

additional 20 min at room temperature. DNA-binding

complexes were resolved by 7% native polyacrylamide

gel electrophoresis with 0.5� TBE (0.045 M Tris-borate/

0.001 M EDTA) with 5 mM Tris/38 mM glycine

running buffer for 90 min at 200 V. The gel was dried,

and complexes were established with excess unlabeled

oligonucleotide.

Measurement of NO

NO scavenging was measured by monitoring 4,5-

diaminofluorescein (DAF-2) by modifying the method

[34]. DAF-2 as a specific NO indicator selectively traps

NO between two amino groups in its molecule and yields

triazolofluorescein, which emits green fluorescence when

excited at 490–515 nm. A stock solution of 1 mg DAF-2

in 0.55 ml dimethyl sulfoxide was stored at �20jC. Aworking solution with 0.5 Ag DAF-2 was diluted with 50

mM phosphate buffer (pH 7.4) purged of nitrogen. The

fluorescence intensity was dependent on the amount of

NO trapped by DAF-2. The fluorescence signal caused

by the reaction of DAF-2 with NO was measured after 10

min using a fluorescence spectrometer FL 500 (Bio-Tek

Instruments, Winooski, VT, USA) at excitation and

emission wavelengths of 490 and 515 nm.

Statistics

The results are presented as means F SE of three

independent triplicate measurements. Statistical signifi-

cance of differences between the untreated control and

the treated groups were determined using one-way anal-

ysis of variance (post hoc test).

RESULTS

Assay of cell viability

The MTT assay was utilized to investigate HHE action

on cell viability. A serum-free medium was selected to

exclude serum albumin binding. Incubation using various

doses of HHE (10–40 AM) showed marked decreases in

cell viability in a dose-dependent manner (Fig. 1). But

cells exposed to HHE for 1 h at concentrations of 10–40

AMshowed no changes in cell viability (Fig. 1). At 30 AM,

Fig. 2. Effects of lipid peroxidation derivative aldehydes on NF-nBactivation in transfected endothelial cells. Cells were grown to 80–90%confluence after being transfected with a reporter plasmid containingpTAL-NF-nB. Cells were treated for 6 h with HHE orMDA and lysed fordetermination of luciferase. Control, untransfected cells; T-control,transfected and untreated cells; HHE, cells transfected with HHE (10AM); MDA, cells transfected with MDA (10 AM); RLU, relative lightunits. Statistical significance: *p < .05 vs. control; #p < .05 vs. T-control.

J. Y. Lee et al.542

the number of nonviable cells increased at 12 h of HHE

exposure, and at 24 h of HHE exposure, nearly 60% of

cells lost viability.

Activation of NF-jB-dependent luciferase induced by

HHE

Free radical-mediated lipid peroxidation can produce

lipid aldehyde products such as MDA as well as HHE.

RS are generated concomitantly during inflammation,

and NF-nB is closely related to the inflammatory re-

sponse and to RS. Thus, we first examined whether these

aldehydes activated NF-nB in endothelial cells. Transient

transfection of a plasmid containing pTAL-NF-nB 5.0

kb, linked to a luciferase reporter construct, illustrated

the transactivation of an NF-nB-dependent reporter geneby HHE and MDA (Fig. 2). MDA did not affect

luciferase activity. In contrast, NF-nB luciferase activity

increased 2-fold compared to that of a transfected cell

exposure to 10 AMHHE for 6 h. The NF-nB transactivity

Fig. 3. Induction of NF-nB activation through InBa degradation by HHavoid the HHE binding affinity of albumin with 30 AM HHE for 0–4 h(0–40 AM) for 1 h. The levels of p65 protein in nuclear extracts (30 Agin were measured in cytosolic extracts (40 Ag/lane). The results are re

increased with HHE treatment at 15 and 20 AM (data not

shown), but a higher dose of MDA (15, 20 AM) did not

affect NF-nB transactivation. Our data showed that HHE

affected NF-nB activity more strongly than MDA using

the same concentrations for both. Therefore, we selected

HHE for the following experiments.

Enhanced NF-jB (p65) translocation and cytosolic IjBadegradation by HHE

We performed Western blot analysis to elucidate the

mechanism that leads to the activation of NF-nB by

HHE (see Fig. 3). The two key steps preceding NF-nBactivation are InB degradation in the cytoplasm and NF-

nB translocation to the nucleus. In addition, the disap-

pearance of InB parallels the stimulation of its phos-

phorylation and subsequent InB degradation. Data

shown in Fig. 3A demonstrate that treatment of endo-

thelial cells with HHE resulted in enhanced p65 protein

in nuclear extracts. The increased expression was de-

tectable within 1 h, continued to 2 h, and then declined

to the untreated control level after 3 h stimulation with

30 AM HHE. When examined after 1 h exposure, the

p65 protein level was increased 3-fold compared to that

of the control (Fig. 3B). Generally, cytosolic activation

of NF-nB requires phosphorylation of InBa protein

before the release by proteolytic degradation from the

complex for translocation of NF-nB to the nucleus.

Examining the disappearance of InBa in cytoplasm, as

shown Fig. 3, InBa expression significantly decreased

with the 1 h exposure to 30 AM HHE, which continued

to 2 h. At 1 h after treatment with HHE, InBadegradation gradually increased in a dose-dependent

manner. These data indicate that HHE induces the

degradation of InB, leading to the nuclear translocation

of the p65 and transactivation of NF-nB-dependent geneexpression.

Induction of NF-jB binding activity

To confirm the effects of HHE on the NF-nB activa-

tion, EMSA was carried out with nuclear proteins.

Treatment of endothelial cells with HHE for different

times led to the activation of the NF-nB transcription

E. (A) The cells were incubated in serum-free medium in order to. (B) The cells were incubated in serum-free medium with HHE/lane) were analyzed by Western blot. The levels of InBa proteinpresentative of three independent experiments.

Fig. 4. Data on the induction of NF-nB binding activity by HHE usingthe gel shift assay. (A) The cells were incubated in serum-free mediumwith HHE for the indicated times. Nuclear fractions were incubatedwith 32P-end-labeled probe containing a binding site for NF-nB. Gelshift assays with NF-nB probes were performed as described underMaterials and Methods. The data presented are representative of at leastthree separate experiments. Lane 1, negative control without nuclearextract; lane 2, treatment with 30 AM HHE for 0 h; lane 3, treatmentwith 30 AM HHE for 0.5 h; lane 4, treatment with 30 AM HHE for 1 h;lane 5, treatment with 30 AMHHE for 2 h; lane 6, treatment with 30 AMHHE for 3 h; lane 7, specific competition with unlabeled NF-nBoligonucleotide. (B) Quantification of the DNA binding activity of NF-nB was performed by densitometric analysis. Statistical significance:*p < 0.05 vs. untreated control.

Fig. 5. Upregulation of iNOS expression induced by HHE. The cellswere incubated in serum-free medium, in order to avoid the HHEbinding affinity of albumin, with 0–30 AM HHE for 24 h. (A) iNOSproteins were analyzed by Western blot. (B) Quantification of iNOSexpression was performed by densitometric analysis. The data presentedare representative of at least three separate experiments. Statisticalsignificance: *p < .05 vs. untreated control.

Induction of iNOS by HNE via NF-nB 543

factor as determined by the increased DNA binding

activity of NF-nB in nuclear fractions. As shown in

Fig. 4, 1 h HHE stimulation significantly enhanced NF-

nB binding activity. Treatment with HHE maximally

increased the NF-nB binding of DNA at 2 h after

stimulation, and activity continued incrementally to 3 h.

These data strongly indicated that HHE enhanced NF-nBactivation.

Upregulation of iNOS expression

Because oxidative stress increases iNOS expression,

we assessed the induction of iNOS by the free radical-

mediated lipid peroxidation by-product, HHE. The iNOS

expression was studied by extracting cellular protein

from HHE-treated cells using Western blot analysis.

Untreated control endothelial cells showed low levels

of constitutive NOS expression, which also has been

shown in other cell systems [35]. It is well known that

NO produced by endothelial (e) NOS under physiolog-

ical conditions plays an essential role in regulation of

vessel tone. However, NO released from iNOS by stimuli

like HHE or uncontrolled conditions like inflammation

can cause a variety of damages. In the present study, the

iNOS protein was detectable in the cellular extract of

untreated cells. After exposure to HHE, the iNOS protein

level showed a marked increase compared to untreated

controls. As shown in Fig. 5, the iNOS protein level

significantly increased after treatment with 20–30 AMHHE for 24 h.

J. Y. Lee et al.544

Inhibitory effects of Bay11-7082 and NAC on

HHE-induced NF-jB activation

Because we observed the induction of NF-nBactivation by HHE, we investigated the ability to

block the induction of NF-nB activation though InBaphosphorylation and consequent degradation using the

specific NF-nB inhibitor Bay 11-7082. Bay 11-7082

interferes with NF-nB by inhibiting InBa phosphory-

lation [25], which was effective at the low dose level

of 2 AM. After pretreatment with 2 AM Bay11-7082

for 30 min, cells were incubated with 30 AM HHE for

an additional 1 h. Cytosolic InB degradation and p65

translocation into the nucleus induced by HHE was

significantly blocked by NF-nB inhibitor (Fig. 6A). To

further investigate whether this inhibitor abolishes

HHE-induced NF-nB activation, EMSA was carried

out with nuclear protein. After preincubation with 2

AM Bay 11-7028 for 30 min, the cells were treated

with 30 AM HHE for an additional 1 h, and NF-nBactivation was determined. As shown in Fig. 6B, Bay

11-7028 significantly blunted the HHE-induced NF-nBbinding of DNA. The suppression of the NF-nBactivation by Bay 11-7028 was confirmed by another

inhibitor of NF-nB, 500 AM NAC, which attenuated

the NF-nB binding activity and p65 translocation into

the nucleus induced by HHE (Fig. 6B).

Inhibition by Bay11-7082 and CAPE of NF-jB luciferase

activity induced by HHE

Because NAC is not only an NF-nB inhibitor, but also

an antioxidant, it may act less specifically on NF-nBinhibition compared to Bay 11-7028 as used in our

system. Therefore, we selected a specific inhibitor for

the translocation of p65 that did not affect InBa degra-

dation [36]. In addition to the inhibition of NF-nBbinding activity as demonstrated in Fig. 7, Bay 11-

7082 and CAPE blunted the induction of HHE-mediated

NF-nB luciferase activity.

Inhibitory effects of Bay11-7082 and NAC on

HHE-induced iNOS expression via NF-jB inactivation

To confirm the role of NF-nB as upstream of expres-

sion of iNOS induced by HHE, we blocked NF-nBactivation with the NF-nB inhibitor Bay 11-7082 (2

AM) and estimated the iNOS expression. Stimulation

with HHE resulted in a profound increase in NF-nBactivity and led to iNOS expression. However, in cells

pretreated with 2 AM Bay 11-7082, enhanced iNOS

protein expression by HHE was attenuated to the level

of the control (Fig. 8). Another NF-nB inhibitor, 500 AMNAC, also decreased the HHE-induced iNOS expression

to a level lower than the untreated control. NAC is

known as a NF-nB inhibitor, as well as a reactive oxygen

species (ROS) inhibitor, that may block iNOS expression

more effectively than Bay 11-7082. Thus, our results

showed that in endothelial cells, HHE can increase iNOS

expression through NF-nB activation.

Inhibitory effects of Bay 11-7082, NAC, and CAPE on

NO levels induced by HHE

NOS catalyzes the breakdown of L-arginine to NO

and citrulline. To verify the possible involvement of NF-

nB activation in the HHE-induced upregulation of iNOS,

NO levels were investigated in endothelial cells. Table 1

shows increases in the NO levels of cells exposed to

HHE for 24 h. At an HHE concentration of 20 AM, the

ON level increased significantly compared to the untreat-

ed controls. To confirm whether HHE triggered NF-nBactivation leading to iNOS upregulation and caused NO

overproduction, we examined the inhibitory effects of

Bay 11-7082, NAC, and CAPE on NO generation in

HHE-treated cells. These inhibitors significantly de-

creased the NO levels induced by HHE to a basal level

(Table 1).

DISCUSSION

It is well documented that the age-associated increase

in the production of reactive aldehydes, such as HNE

and HHE, is due to amplified lipid peroxidation during

aging. HNE and HHE are lipid-derived aldehydes that

have been associated with the etiology of degenerative

disorders including heart disease, atherosclerosis, ische-

mia–reperfusion injury, and diabetes [4–6,37]. Among

the lipid aldehydes, HHE has the most deleterious

effects because of its strong reactivity [13,16]. However,

to date, the molecular mechanism by which HHE acts

on endothelial cell function has not been not fully

explored.

In our present study, we documented that HHE

induces NF-nB transactivation. Our findings further

indicated that the increased activity of NF-nB from

HHE treatment may well be related to the oxidative

status due to a shift in the intracellular redox balance.

Our previous study demonstrated that aging and the age-

related inflammatory processes by NF-nB activation

evolve through an oxidatively disrupted redox balance

[2]. Moreover, recent evidence shows that the activated

NF-nB transactivity involved in the inflammatory pro-

cess underlies many age-related, chronic diseases

[2,38,39]. Thus, during the aging process, dysregulation

of NF-nB activation by HHE could have profound

consequences both in vascular dysfunction and on the

age-related disease status.

New information generated from this current study

on HHE action is owed to this reactive aldehyde’s

characteristic long half-life, easy diffusivity, and perme-

Fig. 6. Inhibition of HHE-induced NF-nB activation. NF-nB activity was determined in HHE-treated cells as described for Fig. 3 or 4.The cells were incubated in serum-free medium with HHE for 1 h after pretreatment for 30 min with Bay 11-7082 or NAC. (A) p65 andInBa gene expression in nucleus or cytosol. (B) Nuclear fractions were incubated with 32P-end-labeled probe containing a binding sitefor NF-nB. Lane 1, negative control without nuclear extract; lane 2, control; lane 3, treatment with 30 AM HHE for 1 h; lane 4, treatmentwith 30 AM HHE for 1 h after pretreatment for 30 min with 2 AM Bay 11-7082; lane 5, treatment with 30 AM HHE for 1 h afterpretreatment for 30 min with 500 AM NAC. Quantification was performed by densitometric analysis. The data presented arerepresentative of at least three separate experiments. Statistical significance: *p < .05 vs. untreated control; #p < .05 vs. 30 AM HHE.

Induction of iNOS by HNE via NF-nB 545

ation through the membrane, which are in contrast to the

activity of free radicals and other reactive species [1].

Thus, we expected reactive, uncharged HHE to migrate

easily through the cytosol from the site of production in

the endothelial membrane, eliciting diverse actions on

cellular functions, including the regulation of gene

transcription.

The binding of lipid reactive products to proteins is a

common occurrence causing vascular damage under

oxidative stress [37]. As far as the physiological concen-

trations of HHE are concerned, van Kuijk et al. reported

that its concentration in LDL of human plasma is at the

level of 13.6 nmol/mg protein [40]. Previous data show

that in human lens epithelial cells exposed to HNE and

Fig. 7. Inhibitory effects of Bay 11-7082, NAC, and CAPE on theluciferase activity of NF-nB induced by HHE. Luciferase activity ofNF-nB was determined as described for Fig. 2. The cells werepretreated with inhibitors for 30 min and treated with HHE for 6 h.Control, untransfected cells; T-control, transfected and untreated cells;HHE, cells transfected with HHE (10 AM); HHE+Bay, cells transfectedwith HHE (10 AM) and Bay 11-7082 (2 AM); HHE+NAC, cellstransfected with HHE (10 AM) and NAC (500 AM); HHE+CAPE, cellstransfected with HHE (10 AM) and CAPE (10 AM); RLU, relative lightunits. Statistical significance: *p < .05 vs. control; #p < .05 vs. T-control.

Fig. 8. Inhibition by NF-nB inhibitors of HHE-induced iNOSexpression. (A) The cells were incubated in serum-free medium for24 h with HHE after pretreatment for 30 min with Bay 11-7082 orNAC. Lane 1, untreated control; lane 2, 30 AM HHE; lane 3, 30 AMHHE + 2 AM Bay 11-7082; lane 4, 30 AM HHE + 500 AM NAC. Thedata presented are representative of at least three separate experiments.(B) Quantification of the iNOS expression was performed bydensitometric analysis. Statistical significance: *p < .05 vs. untreatedcontrol; #p < .05 vs. 30 AM HHE.

Table 1. Protective Effects of Inhibitors of HHE-InducedNO Production

Treatment (AM) NO level (fluorescence

intensity/mg protein)

HHE (0) 814 F 29HHE (10) 972 F 51HHE (20) 1227 F 72*HHE (30) 1969 F 98*HHE (30) + Bay (2) 920 F 35**HHE (30) + NAC (500) 723 F 23**HHE (30) + CAPE (10) 810 F 62**

Bay = Bay 11-7082, NAC = N-acetyl cysteine, CAPE = caffeic acid

methyl ester. The results are presented as means F SE of three

individual experiments. One-factor ANOVA was conducted to analyze

significant differences among untreated control and treated groups.

Differences between the means of individual groups were assessed by

the Fischer Protected LSD post hoc test. Values of p < .05 were

considered statistically significant.*p < .05 vs. untreated control.**p < .05 vs. 30 AM HHE.

J. Y. Lee et al.546

HHE for 24 h, the LD50 was 30 and 50 AM, respectively

[13]. In human venous plasma, normal HNE levels are

estimated to be between 0.3 and 1 AM [41]. However,

under pathological conditions, concentrations of HNE

and HHE can increase significantly and accumulate in

cellular membranes at concentrations up to 5 mM in

response to oxidative insult [1,42]. Thus, the HHE

concentration of 30 AM used in the current experiments

is within the range of physiologically permissible levels.

The production of endothelial ROS/RNS plays an

important role in the NF-nB action that leads to the

enhancement of proinflammatory genes, including

COX-2, iNOS, and cytokines, during aging [2,43,44].

What we were able to show in the current study is

evidence that HHE can elicit NF-nB activation by the

enhanced expression of iNOS. Although many studies

have suggested that iNOS gene expression is regulated

by the NF-nB transcription factor, to date, HHE-related

changes in NF-nB activity and the role of NF-nB in the

HHE-related upregulation of endothelial iNOS have not

been demonstrated.

NO produced by eNOS under physiological condi-

tions plays an essential role in the regulation of vessel

tone [35]. However, NO released from iNOS under

stimulation, such as in the present study from HHE or

under inflammatory conditions, is significantly elevated

to form a potent peroxynitrite by combining with super-

oxide [45].

We previously proposed inflammation and related

proinflammatory processes as major underlying causes

of aging-related chronic disease processes based mainly

on common observations of oxidatively activated key

proinflammatory transcription factors [see review, 44].

The significance of NF-nB activation in inflammation

Induction of iNOS by HNE via NF-nB 547

and vascular changes is 2-fold: (1) NF-nB occupies a

center position in the regulation of expression of several

major proinflammatory proteins, including interleukin

(IL)-1, IL-8, and tumor necrosis factor a [46,47]; (2)

NF-nB modulates the activation of COX-2 and iNOS,

which play major roles in the modulation of normal

vascular function and inflammatory pathogenesis

[23,29,38]. The ability of inflammatory stimuli to induce

iNOS, thereby generating large amounts of NO, strongly

implicates this isoform as a major participant in causing

chronic inflammatory diseases. It is worth pointing out

that our current findings are generated from a YPEN-1

cell system that does not consist of normal primary

cultured cells, thus further confirmatory work may be

needed.

In conclusion, we found iNOS expression in the

endothelial cell through the transcriptional regulation of

NF-nB activation due to InB degradation. We expect the

upregulation of NF-nB and iNOS by reactive aldehydes

highlights the importance of the endogenous lipoperox-

idative process in gene regulation and aging signal

transduction.

Acknowledgment—This work was supported by the Korean ResearchFoundation under Grant KRF-99-0005-F00030/F00037.

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