Review article

Antioxidant and prooxidant mechanisms in the regulation

of redox(y)-sensitive transcription factors

John J. Haddad *

Severinghaus-Radiometer Research Laboratories, Molecular Neuroscience Research Division, Department of Anesthesia and Perioperative Care,

University of California at San Francisco, School of Medicine, Medical Sciences Building S-261, 513 Parnassus Avenue, San Francisco,

CA 94143-0542, USA

Received 2 April 2002; accepted 13 May 2002

Abstract

A progressive rise of oxidative stress due to the altered reduction–oxidation (redox) homeostasis appears to be one of the hallmarks of the

processes that regulate gene transcription in physiology and pathophysiology. Reactive oxygen (ROS) and nitrogen (RNS) species serve as

signaling messengers for the evolution and perpetuation of the inflammatory process that is often associated with the condition of oxidative

stress, which involves genetic regulation. Changes in the pattern of gene expression through ROS/RNS-sensitive regulatory transcription

factors are crucial components of the machinery that determines cellular responses to oxidative/redox conditions. Transcription factors that

are directly influenced by reactive species and pro-inflammatory signals include nuclear factor-nB (NF-nB) and hypoxia-inducible factor-1a

(HIF-1a). Here, I describe the basic components of the intracellular oxidative/redox control machinery and its crucial regulation of oxygen-

and redox-sensitive transcription factors such as NF-nB and HIF-1a.

D 2002 Elsevier Science Inc. All rights reserved.

Keywords: Antioxidant; HIF-1a; NF-nB; Oxygen; Redox; Transcription factors

1. Introduction

Molecular oxygen is an important environmental and

developmental signal that regulates cellular energetics,

growth and differentiation [1–4]. Despite its central role

in nearly all higher life processes, the molecular mecha-

nisms for sensing oxygen levels and the pathways involved

in transducing this information remain largely obscure [1,3].

Oxygen, a gaseous element with colorless, odorless and

tasteless appearance, is the most abundant element on planet

earth, making up about 20% by volume of the atmosphere at

sea level, about 50% of the material of the earth’s surface

and about 90% of water. Biologically, oxygen is necessary

for sustaining the life processes of nearly all living organ-

isms and, chemically, for most forms of combustion. It

readily forms compounds with nearly all other known

elements, except the inert gases, and it is used in blast

furnaces, steel manufacture, chemical synthesis, in resusci-

tation and for many other industrial purposes. Oxygen, then,

is an essential molecule for all aerobic life forms; however,

oxygen plays univalent roles: while oxygen is indispensable

for the cell to obtain the essential chemical energy as a form

0898-6568/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved.

PII: S0898 -6568 (02 )00053 -0

Abbreviations: NAC, N-acetyl-L-cysteine; AP-1, activating protein-1;

ASK, apoptosis signal-inducing kinase; ATF, activating transcription factor;

ATP, Adenosine triphosphate; AOP, anti-oxidative potential; ARE, antiox-

idant responsive element; ARDS, adults respiratory distress syndrome;

ARNT, aryl hydrocarbon nuclear translocator; bHLH, basic helix– loop–

helix; BCNU, 1,3-bis-(2-chloroethyl)-1-nitrosourea; BPD, bronchopulmo-

nary dysplasia; BSO, L-buthionine-(S,R)-sulfoximine; BHA, butylated

hydroxyanisole; Cd, cadmium; CREB, cAMP-responsive element binding

protein; CF, cystic fibrosis; JNK, c-Jun N-terminal kinase; DPI, diphenylene

iodonium; EPO, erythropoietin; GLUT, glucose transporter; GSH, L-g-

glutamyl-L-cysteinyl-glycine; GRX, Glutaredoxin; GSSG, glutathione

oxidized disulfide; g-GCS, g-glutamylcysteine synthetase; HO, heme

oxygenase; H2O2, hydrogen peroxide; OH, hydroxyl radical; HOCl,

hypochlorous acid; HIF-1a, hypoxia-inducible factor-1a; InB, inhibitoryprotein; IKK, inhibitory nB kinase; IL, interleukin; LPS, lipopolysacchar-

ide-endotoxin; MAPK, mitogen-activated protein kinase; MT-1, metal-

lothionein-1; NF-nB, nuclear factor nB; NIK, NF-nB inducing kinase;

NLS, nuclear localization signal; NO, nitric oxide; PKA, protein kinase A;

PAS, Per-ARNT-Sim; PDTC, pyrrolidine dithiocarbamate; Redox, reduc-

tion–oxidation; RHD, Rel homology domain; RNS, reactive nitrogen

species; ROS, reactive oxygen species; 1O2, singlet oxygen; O2�S

,

superoxide anion; SOD, superoxide dismutase; TRX, thioredoxin; TNF-a,

tumor necrosis factor-a; TNFR, TNF receptor; VEGF, vascular endothelial

growth factor.* Tel.: +1-415-476-8984; fax: +1-415-476-8841.

E-mail address: haddadj@anesthesia.ucsf.edu (J.J. Haddad).

www.elsevier.com/locate/cellsig

Cellular Signalling 14 (2002) 879–897

of ATP, it is often transformed into highly reactive forms,

radical oxygen species (ROS), which are often toxic for the

cell [1,3,5–18]. In order to defend themselves from the

cytotoxic actions of ROS and other free radicals, cells have

acquired multiplicity in endogenous antioxidant systems

during the long evolutionary periods. These defense mech-

anisms include reduction–oxidation (redox) enzymatic sys-

tems such as glutaredoxin and thioredoxin [1–3,19–37].

Studies having the nature of cell biology and molecular

biochemistry have revealed that these molecules are also

involved in cell signaling [1–3,6,15–17,28,33,38–54]. The

term ‘oxidative regulation’ has thus been proposed to

indicate the active role of oxide-reductive modifications of

proteins in regulating their functions. Oxide-reductive reac-

tions of bio-molecules, mostly proteins, formerly considered

as ‘oxidative stress,’ are now considered as ‘signals’ and

contain biological information that is necessary for main-

taining cellular homeostasis [1–3,6,24,28,35,39,55–73].

Altering gene expression is the most fundamental and

effective way for a cell to respond to extracellular signals

and/or changes in its environment, in both the short term and

long term. In the short term, transcription factors are

involved in mediating responses to growth factors and a

variety of other extracellular signals In contrast, the long-

term control of gene expression induced by growth factors

and the changes in gene expression, which occur during

development, are generally (with few exceptions) irreversi-

ble [1,6,17,19,21,24,28,35,50,60,74–105]. During develop-

ment, the expression of specific sets of genes is regulated

spatially (by position/morpho-genetic gradients) and tempo-

rally. Regulation of the signaling responses is governed at the

genetic level by transcription factors that bind to control

regions of target genes and alter their expression. Tran-

scription factors are endogenous substances, usually pro-

teins, which are effective in the initiation, stimulation, or

termination of the genetic transcription process, while in the

cytoplasm, the transcription factor is incapable of promoting

transcription. A signaling event, such as a change of the state

of phosphorylation, occurs, which results in protein subunit

translocation into the nucleus. Transcription is a process in

which one DNA strand is used as a template to synthesize a

complementary RNA. Signal transduction, therefore, in-

volves complex interactions of multiple cellular pathways

[1,2]. In particular, interest in reduction–oxidation/oxygen

(redox{y})-sensitive transcription factors has gained an

overwhelming backlog of interest momentum over the years

ever since the onset of the burgeoning field of free radical

research and oxidative stress. The reason for this is that

redox(y)-sensitive transcription factors are often associated

with the development and progression of many human

disease states; therefore, their ultimate regulation bears

potential therapeutic intervention for possible clinical appli-

cations [9,11,12,17,22,25,37,38,42,47,54,58,69,91,106–

120]. In this review, I will focus on elaborating a compre-

hensive overview of the current understanding of redox/

oxidative mechanisms mediating the regulation of key tran-

scription factors, particularly nuclear factor-nB (NF-nB) andhypoxia-inducible factor-1a (HIF-1a), which regulate a

plethora of cellular functions that span the range from anoxia

and hypoxia to extreme hyperoxia and oxidative stress, both

in physiologic and pathophysiologic conditions.

2. Reduction–oxidation concepts: the paradigm of

oxidative stress

The earliest view of the redox concept is that of the

addition of oxygen molecule (oxidation) to form an

oxidant, or removal of oxygen (reduction) to form a re-

ductant [3,24]. For example, in the burning of hydrogen

(2H2 +O2! 2H2O), the hydrogen is oxidized and the

oxygen is reduced. The combination of nitrogen and

oxygen, which occurs at high temperatures, follows the

same pattern (N2 +O2! 2NO). This formation of NO

oxidizes the nitrogen and reduces the oxygen. In some

reactions, the oxidation process is most prominent. For

example, in the burning of methane (CH4 + 2O2!CO2 +

2H2O), both carbon and hydrogen are oxidized (gain oxy-

gen). The accompanying reduction of oxygen is perhaps

easier to see when the process of reduction is described as the

gaining of hydrogen. On the other hand, the reaction of lead

dioxide at high temperatures appears to be just reduction

(2PbO2! 2PbO +O2). The reduction of PbO2 is clear, but

the associated oxidation of oxygen is easier to see when the

process of oxidation is described as the losing of electrons.

Furthermore, an alternative approach is to describe oxidation

as the loss of hydrogen and reduction as the gaining of

hydrogen. This has an advantage in describing the burning of

methane, for instance. With this approach, it is clear that the

carbon is oxidized (loses all four hydrogens) and that part of

the oxygen is reduced (gains hydrogen). Another alternative

view is to describe oxidation as the losing of electrons and

reduction as the gaining of electrons [3]. Redox concepts are

depicted in a hypothetical overview in Fig. 1.

Today, the idea of free radicals gets a new dimension. Our

human body is more under attack from a free radical-invoked

Fig. 1. An overview of the basic mechanisms mediating reduction–

oxidation (redox) concepts in chemico-biologic interactions.

J.J. Haddad / Cellular Signalling 14 (2002) 879–897880

condition generally referred to as ‘oxidative stress’ [1,5,11–

13,16,17,24,28,33,34,45,50,55,59,94,95,111,114,115,117,1-

21–127]. Each human organ and each human cell is

influenced by oxidative stress, which is separated into

internal (inflammation, autoimmune reactions, dysregula-

tion of metabolism, ischemia) and external (microbiological

organism, electromagnetic radiation, mechanical-, thermal-

and chemical-induced stress) stresses. Oxidative damage

defines the consequences of a mismatch between the pro-

duction of the reactive oxygen and nitrogen species (ROS/

RNS) and the ability to defend against them. For the most

part, ROS/RNS originate in the body; they are numerous

and include free radicals such as superoxide (O2� ), nitric

oxide (NO) and hydroxyl ion (OH), as well as the oxygen-

derived species such as singlet oxygen (1O2), hydrogen

peroxide (H2O2) and hypochlorous acid (HOCl) (Fig. 2)

[1,3,4,7,16,20,24,42,78]. Major sources of ROS/RNS

include, but are not exclusive to, mitochondrial oxidative

metabolism, phospholipid metabolism and proteolysis. Bio-

logical systems are protected from the threat of oxidative

assault by a diversity of mechanisms designed to suppress

pernicious oxidative pathways. Raised against the challenges

are an extensive and highly effective array of protective

agents and defense antioxidant mechanisms. These comprise

numerous small molecular weight antioxidants to forestall

initiation of oxidative damage and/or limit its propagation,

enzymes that convert and detoxify ROS/RNS, enzymes to

repair oxidative damage when it occurs and mechanisms to

route damaged molecules for destruction and replacement

[10,17,18,20,22,25,26,28,33,54,79,87,119,120,127–138].

For mitochondria where reactive intermediates inevitably

leak from the electron transport chain to cause local damage,

rapid turnover and replacement of the organelle is crucially

recognized. In fact, the need for rapid regeneration of

mitochondria is often used to explain its independent store

of DNA; lastly, outer segments have a turnover mechanism

based on disc renewal and shedding to supplement local

repair [1,2,4,24,139]. Antioxidant processes usually work by

direct scavenging of the initiating prooxidant species. Each

tissue, for instance, has an anti-oxidative potential (AOP),

which is determined by the balance and those exerting an

enzymatic and non-enzymatic antioxidants to indicate a need

for such protection. A healthy cell is one in which the

antioxidant systems effectively keep the level of prooxidants

below a critical, non-pernicious threshold. ROS/RNS rad-

ical pathways and their enzymatic dismutation are shown

in Fig. 3.

3. Oxygen radicals and redox regulation of NF-KB

signaling

3.1. The Rel/NF-jB family: an overview

To accommodate an ever-changing microenvironment,

cells adjust the pattern of gene expression by adaptive

regulation of a host of transcription factors, which bind their

respective cognate sites in the regulatory elements of targeted

genes (Fig. 4) [1,2,91,140]. The recognition of ROS/RNS

and redox-mediated protein modifications as transducing

signals has opened up a new field of cell regulation and

provided a novel way of controlling disease processes. One

such approach has been proven feasible for gene expression

governed by the transcription factor NF-nB. The nature of

the redox/oxidative mechanisms mediating the regulation of

NF-nB, as a typical example of tightly controlled cellular

signaling, will be thoroughly discussed here.Fig. 2. The oxygen triangle and the various associated free radicals

emanating from its corners.

Fig. 3. Selective dismutation of reactive oxygen (ROS) and nitrogen (RNS)

species, representing a schematic model of the pathways leading to the

generation of ROS/RNS. A number of major cellular enzymes that defend

against oxidative stress have been conserved through evolution. Superoxide

(O2� ) anion is metabolized via the dismutation reaction 2O2

� +

2H + !O2 +H2O2, which is catalyzed by superoxide oxidoreductase

dismutase (SOD), a cytoplasmic enzyme that is constitutively expressed,

and a mitochondrial enzyme that is induced in response to oxidant stress.

The H2O2 produced by the dismutation of O2� is converted by one pathway

to H2O and O2 by catalase (CAT) in peroxisomes and by glutathione

peroxidase (GSH-PX) in the cytoplasm, at the expense of reduced

glutathione (GSH), leading to the formation of oxidized glutathione

disulphide (GSSG) that is recycled back to GSH by glutathione reductase

(GSSG-RD). H2O2 could be further converted by another pathway

involving iron into hydroxyl radical (OH), an injurious ROS causing

cellular damage. This iron-catalyzed reaction, known as the Fenton-like

reaction, is impeded by the iron chelator desferrioxamine (DSF), which is

also capable of neutralizing the toxicity of OH.

J.J. Haddad / Cellular Signalling 14 (2002) 879–897 881

Although the transcription factor NF-nB has been orig-

inally recognized in regulating gene expression in B-cell

lymphocytes [49], subsequent investigations have demon-

strated that it is one member of a ubiquitously expressed

family of Rel-related transcription factors that serve as

critical regulators of many genes, including those of pro-

inflammatory cytokines [6,21,28,40,55,63,65,74,76,86–

89,91,94,121,133,134,140–163]. NF-nB comprises the Rel

family of inducible transcription factors that are key media-

tors in regulating the progression of the inflammatory process

[2,35,39,40,44,63,65,82–84,87,91,94,95,111,132,141,164–

166]. Therefore, activation and regulation of the NF-nB/Reltranscription family, via nuclear translocation of cytoplas-

mic entities and complexes, play a central role in the

evolution of inflammation through the regulation of genes

essentially involved in encoding pro-inflammatory cytokines

and other inflammatory mediators [167]. The NF-nB/Relfamily includes five members: NF-nB1 (p50/p105 {p50

precursor}), NF-nB2 (p52/p100 {p52 precursor}), RelA

(p65), RelB (p68) and c-Rel (p75) [63,92,148,158,168–

172]. Despite the ability of most Rel members (with the

exception of p68) to homodimerize, as well as to form

heterodimers, with each other, the most prevalent activated

form of NF-nB is the heterodimer p50–p65, which possesses

the transactivity domains necessary for gene regulation

(Fig. 5). The NF-nB members contain a Rel homology

domain (RHD), which is responsible for dimer formation,

nuclear translocation, sequence-specific consensus DNA

recognition and interaction with inhibitory-nB (InB) pro-

teins, which are the cytosolic inhibitors of NF-nB. The

translocation and activation of NF-nB in response to various

stimuli are sequentially organized at the molecular level. In

resting, unstimulated cells, NF-nB resides in the cytoplasm as

an inactive NF-nB/InB complex, a mechanism that hinders

the recognition of the nuclear localization signal (NLS) by

the nuclear import machinery, thereby retaining the NF-nBcomplex within the cytosol. In its inactive state, the hetero-

dimeric NF-nB, which is mainly composed of two subunits,

p50 (NF-nB1) and p65 (RelA), is present in the cytoplasm

associated with InB. Upon stimulation, such as with cyto-

kines and lipopolysaccharide-endotoxin (LPS), derived from

the cell wall of Gram-negative bacteria, InB-a, the major

cytosolic inhibitor of NF-nB, undergoes phosphorylation on

serine/threonine residues, ubiquitination and subsequent pro-

teolytic degradation, thereby unmasking the NLS on p65 and

allowing nuclear translocation of the complex (Fig. 5). This

sequential propagation of signaling ultimately results in the

release of NF-nB subunits from InB-a inhibitor, allowing

translocation and promotion of gene transcription. Signals

emanating from membrane receptors, such as those for IL-1

and TNF-a, activate members of the MEKK-related family,

including NF-nB-inducing kinase (NIK) and MEKK1, both

of which are involved in the activation of InB kinases, IKK1

and IKK2, components of the IKK signalsome [50,63,

78,87,112,143,145,148,150,173–177]. These kinases phos-

phorylate members of the InB family, including InB-a, themajor cytosolic inhibitor of NF-nB, at specific serines withintheir amino termini, thereby leading to site-specific ubiquiti-

nation and degradation by the proteasome (Fig. 5). This

sequential trajectory culminating in the inducible degradation

of InB, which occurs through consecutive steps of phosphor-ylation and ubiquitination, allows freeing of the NF-nBcomplex, which translocates to the nucleus to bind specific

nB moieties and initiate gene transcription (Fig. 6).

Rel/NF-nB transcription factors are a family of structur-

ally related eukaryotic transcription factors that are involved

in the control of a vast array of processes, such as immune

and inflammatory responses, developmental processes, cel-

lular growth and programmed cell death (apoptosis). In

addition, these factors are active in a number of disease

states, including cancer, arthritis, inflammation, asthma,

neurodegenerative diseases and cardiovascular abnormal-

ities [10,17,25,38,44,55,58,69,91,95,112,113,166,178]. The

immunoregulatory approach aimed at targeting the NF-nB

Fig. 5. Transcriptional regulation of NF-nB and the structures of the Rel

family transcription factors. Shown are the generalized structures of the two

classes of Rel transcription factors. All have a conserved DNA-binding/

dimerization domain called the Rel homology (RH) domain, which also has

sequences important for nuclear localization (N) and InB inhibitor binding.

Class I proteins have additional inserted sequences in the RH domain. The

C-terminal halves of the class I Rel proteins have ankyrin repeat-containing

inhibitory domains, which can be removed by proteasome-mediated

proteolysis (Protease). The C-terminal halves of the class II Rel proteins

have transcriptional activation domains.

Fig. 4. Gene transcriptional overview implicating selective transcription

factor–DNA complex interactions.

J.J. Haddad / Cellular Signalling 14 (2002) 879–897882

signaling pathway, therefore, remains of particular interest.

Since NF-nB regulates host inflammatory and immune

responses by increasing the expression of specific genes

and enzymes whose products contribute to the pathogenesis

of the inflammatory process, selective modulation of this

transcription factor bears a typical therapeutic approach for

the control and regulation of inflammatory-associated dis-

eases. Unfortunately, due to convergence of more than one

mechanism upon the onset and progression of the inflam-

matory process, which regulates NF-nB signaling, it has

been extremely difficult solely to target this pathway with-

out affecting other cellular functions. The myriad of genes

directly targeted by NF-nB is given in Table 1.

3.2. Oxidative stress-mediated regulation of NF-jB

What are the signaling cofactors that trigger and mediate

NF-nB signaling cascades? Not much is known about what

exactly happens immediately downstream of cell surface

receptors. The expression of genes in response to trans-

ducing signals from surface receptors is predominantly

determined by the conditions of the cell microenvironment.

Prime examples of such regulation are found in embryonic

development of all multicellular organisms [24,25,179]. The

naturally occurring regulating agents, for example, interact

with specific receptors, which subsequently transduce a

signal onto the nucleus for the regulation of gene expression

and activation. The putative oxygen sensor responds to

dynamic variation in pO2. Upon ligand binding, this mem-

brane-bound receptor transduces intracellular chemical/

redox signals that relay messages for the regulation of gene

expression, a phenomenon mainly involving the activation

of transcription factors (Fig. 6) [6,17,28,50,149,179,180].

NF-nB is among the most important transcription factors

shown to respond directly to oxidative stress [6,8,15,28,55,

68,70,73,74,76,77,118,121,160,174,179,181–183]. Antiox-

idants, for instance, have been reported to block NF-nBactivation in certain cell types, leading to the hypothesis

that the activation of this transcription factor is mediated

by ROS in response to specific stimuli [10,28,35,36,50,70,

71,95,133,134,136,137,140,144,145,153,159,173,177,184].

The involvement of ROS is postulated to regulate and

modulate the activity of the upstream kinases that converge

onto the NF-nB signaling activation pathway. In this respect,

wide arrays of antioxidants that can detoxify ROS/RNS have

been reported to suppress the activation of NF-nB, suggest-ing a key role for reactive species in NF-nB signaling. For

instance, hepatocarcinogen 2-acetylaminofluorene treat-

ments led to the increase of intracellular ROS, which caused

Table 1

Direct NF-nB target genes

Cytokines/Growth factor

Interleukin (IL)-1a, IL-1h, IL-2, IL-3, IL-6, IL-8, IL-12, Tumor Necrosis

Factor (TNF)-a, Lymphotoxin (LT)-a, Interferon (IFN)-h, GranulocyteColony-Stimulating Factor (G-CSF), Macrophage Colony-Stimulating

Factor (M-CSF), Granulocyte-Macrophage Colony-Stimulating Factor

(GM-CSF)

Cytokine receptors

IL-2 Receptor a-Chain (IL-2Ra)

Stress proteins

Serum Amyloid A Protein (SAA), complement factors B, C3 and C4, a1-

Acid Glycoprotein

Adhesion molecules

Intracellular Adhesion Molecule 1 (ICAM-1), Vascular Cell Adhesion

Molecule 1 (VCAM-1), Mucosal Addressin Cell Adhesion Molecule 1

(MAdCAM-1), E-Selectin

Immunoregulatory molecules

Immunoglobulin n Light Chain (Ign), Major Histocompatibility complex

(MHC class I and II), T-Cell Receptor (TCRa and h), h2-Microglobulin,

Invariant Chain (Ii), Transporter Associated with Antigen Processing

(TAP-1), Proteasome Subunit (LMP-2), Inducible Nitric Oxide Synthase

(iNOS), Inhibitory nB (InB), p53

Fig. 6. Rel/NF-nB signal transduction. Various signals converge on

activation of the InB kinase (IKK) complex. IKK then phosphorylates InBat two N-terminal serines, which signals it for ubiquitination and proteolysis.

Freed NF-nB (p50-RelA) enters the nucleus and activates gene expression.

The activation of NF-nB is thought to be part of a stress response as it is

activated by a variety of stimuli that include growth factors, cytokines,

lymphokines, UV, pharmacological agents, and stress. In its inactive form,

NF-nB is sequestered in the cytoplasm, bound by members of the InB family

of inhibitor proteins, which include InB-a, InB-h, InB-g and InB-q. Thevarious stimuli that activate NF-nB cause phosphorylation of InB, which is

followed by its ubiquitination and subsequent degradation. This results in

the exposure of the nuclear localization signals (NLS) on NF-nB subunits

and the subsequent translocation of the molecule to the nucleus. In the

nucleus, NF-nB binds with a consensus sequence (5V-GGGACTTTCC-3V) ofvarious genes and thus activates their transcription.

J.J. Haddad / Cellular Signalling 14 (2002) 879–897 883

the activation of IKK kinases, the degradation of InB-h and

the accumulation of NF-nB in the nuclear compartment

[147]. Similarly, a-phenyl-tert-butylnitrone, an effective

spin-trapping agent that reacts with and stabilizes free radical

species, has been shown to inhibit pancreatic h cell death and

the development of insulin-dependent diabetes mellitus in an

NF-nB-dependent pathway [35,133,134,153]. Furthermore,

superoxide dismutase (SOD) expression has negative effects

on the activation of NF-nB in transient focal cerebral

ischemia, indicating the involvement of specific ROS

[173]. Of interest, treatment of mammalian cells with H2O2

induced the nuclear translocation of NF-nB and its binding to

nB DNA sequences present in the promoter region of

numerous genes. The role of selenium in NF-nB activation

was analysed in human T47D cells over-expressing the

seleno-dependent detoxifying enzyme glutathione peroxi-

dase [182]. Following exposure to H2O2, these cells showed

a seleno-dependent decreased accumulation of intracellular

ROS and NF-nB activation. This phenomenon was corre-

lated with an inhibition of the nuclear translocation of NF-nB(p50 subunit) and with an absence of InB-a degradation. It

was also reported that the half-life of InB-a in untreated cells

was increased two-fold by the overexpression of the active

glutathione peroxidase. These results suggest that selenium

is a key element that, through its modulation of glutathione

peroxidase activity, can inhibit NF-nB and can up-regulate

cytosolic InB-a normal half-life [182]. In addition, an

impaired pulmonary NF-nB activation has been observed

in response to LPS in NADPH oxidase-deficient mice [156].

Of note, xanthine oxidase-derived ROS can activate NF-nB[185] and cellular enrichment with polyunsaturated fatty

acids can induce the development of oxidative stress con-

dition and the activity of activating protein-1 (AP-1) and NF-

nB [72]. Furthermore, hyperoxia has been reported to up-

regulate the nitric oxide (NO)-sensitive pathway in vitro and

similarly activate AP-1 and NF-nB, suggesting a role for

RNS [186].

Because NF-nB can be rapidly induced in a variety of

cell types by a diverse set of seemingly unrelated agents, it

has been proposed that agents activating this transcription

factor do so by increasing a minimum intracellular effective

oxidative stress threshold [14,55,73,96,126,160,186]. A

novel recent study by Blackwell et al. [76] has highlighted

the significance of oxidative stress in regulating NF-nB.Although in vitro data has linked ROS to the activation of

NF-nB, little information exists regarding this relationship

in human disease. It was hypothesized that bone marrow

transplantation (BMT), for instance, would impart a degree

of oxidative stress that might lead to the in vivo activation of

NF-nB. Because NF-nB regulates transcription of proin-

flammatory mediators, it was reasoned that the activation of

NF-nB might contribute to the development of transplant-

related complications. To evaluate NF-nB activation in

humans, Blackwell and colleagues measured NF-nBDNA-binding activity in nuclear extracts of bronchoalveolar

lavage (BAL) cells obtained before and after allogeneic

BMT. Changes in BAL cells NF-nB activation were com-

pared with changes in urinary F2-isoprostane concentration,

an indicator of in vivo free radical-catalyzed lipid perox-

idation. Although the extent of the in vivo lipid peroxidation

has substantial inter-individual variability over time, it was

found that there was a strong correlation between the pre/

post-BMT ratio of urinary isoprostane concentrations and

pre/post-BMT ratio of NF-nB binding activity in BAL cells.

Although limited by the small number of patients studied,

this data closely linked oxidative stress to NF-nB activation

in human alveolar macrophages following BMT. It is

possible that such interactions may contribute to the clinical

course after BMT by affecting the transcription of proin-

flammatory genes [76]. On the oxidative mechanisms that

mediate the regulation of NF-nB, the effect of pyrrolidine

dithiocarbamate (PDTC), which potently blocks the activa-

tion of NF-nB in serum-exposed conditions by reducing

ROS [15,183], was explored on the activation of MAPKs

[144]. PDTC transiently increased the phosphotransferase

activity of c-Jun N-terminal kinase-1 (JNK-1), which in turn

activated the transcriptional activity of AP-1. The activation

of JNK was completely decreased in dominant negative

JNK-1 transfected cells and the PDTC-induced cell death

was attenuated in these cells. In addition, AP-1 activation

was decreased in the JNK-1 transfected cells, compared

with vector-transfected cells. The NF-nB inhibitor also

transiently activated MAPKp38 but its inhibition did not

affect the regulatory effect on PDTC-induced cell death,

suggesting that oxidative stress-induced cell death is medi-

ated by MAPKJNK and not by MAPKp38. Furthermore, it

has been reported that the tumor necrosis factor receptor

(TNFR) is implicated in ROS-mediated regulation of NF-nBand its involvement in creating balance between pro- and

anti-apoptotic signaling cofactors governing cell fate in

oxidative stress [56,78]. In accordance with the aforemen-

tioned observations, a cytokine-responsive upstream InBkinase has been found to initiate a ROS-dependent activa-

tion of the NF-nB signaling pathway, suggesting the

involvement of selective kinases in cytokine-mediated NF-

nB regulation [148,177]. Similarly, the involvement of NF-

nB in cardiomyocyte hypertrophy requires the activation of

apoptosis signal-regulating kinase-1 (ASK-1) in a G-pro-

tein-dependent pathway, which was postulated to allow the

induction of intracellular ROS [165]. Of note, it has been

reported that the cooperativity between oxidants and TNF-a

in the activation of NF-nB requires the involvement of a

Ras/Rac/MAPK-dependent mechanism [161,174,181,187].

Moreover, the mitochondria-derived ROS have been

observed as crucial mediators in the regulation of NF-nB[188]. For instance, it was found that the activation of NF-

nB by TNF-a was blocked by rotenone or amytal, inhibitors

of complex I of the mitochondrial respiratory chain [189].

On the other hand, antimycin A, an inhibitor of complex III,

enhanced TNF-a activation of NF-nB, indicating a key role

of mitochondria-derived ROS in mediating NF-nB signaling

[81,189]. Furthermore, g-glutamyl-transpeptidase (GGT), a

J.J. Haddad / Cellular Signalling 14 (2002) 879–897884

key enzyme implicated in the homeostasis of intracellular

reduced GSH, and hence in the regulation of the cellular

redox state, has been implicated in the extracellular cleavage

of GSH and the generation of ROS. Using a model cell line,

the V79 GGT, which highly expresses a human GGT

transgene, GGT-induced oxidant stress was shown to mod-

ulate intracellular transcription factors. GGT-dependent

ROS production induced the NF-nB-binding and transacti-

vation activities [19]. This induction mimicked the one

observed by H2O2 and was inhibited by catalase, suggesting

the involvement of H2O2 in the NF-nB activation.

However, the model of ROS/RNS (oxidative stress) as

exclusive messengers in the regulation of the NF-nB signal-

ing pathway cannot be universally accounted for (Fig. 7)

[16,24,121,142,175,190–192]. For instance, Ginis and col-

leagues [193] reported that ROS could act synergistically

with TNF-a in causing cytotoxicity via the inhibition of a

cytoprotective branch of TNF-a signaling pathways, which

starts with NF-nB activation. Furthermore, addition of

glucosamine to rat chondrocytes treated with IL-1h or with

ROS decreased the activation of NF-nB, but not that of AP-1. Of note, it has been recently reported that irreversible

inhibition of g-glutamylcysteine synthetase (g-GCS), the

rate-limiting enzyme in the biosynthesis of glutathione

(GSH), an antioxidant thiol, was associated with the aug-

mentation of a pro-inflammatory signal in a ROS-sensitive

manner despite the observation that the InB-a/NF-nB sig-

naling pathway was down-regulated [32,38,40,67]. The

intriguing ability of g-GCS inhibition to block the trans-

location/activation of NF-nB and up-regulate the presum-

ably downstream cytokine pathway remains of particular

interest. It was reported that L-buthionine-(S,R)-sulfoximine

(BSO), an inhibitor of g-GCS, suppressed the oxyexcitation

(DpO2)-dependent nuclear localization of RelA (p65), the

major transactivating member of the Rel family, and sub-

sequently suppressed NF-nB activation [32,38,67]. How-

ever, in additional studies, BSO was also shown to be

capable of inducing intracellular accumulation of ROS,

particularly OH [6,7,28,29,38,83]. Taken together, these

data argue for ROS as potential second messengers for

cytokine biosynthesis; however, ROS might not be favor-

ably universal messengers in the activation of NF-nB.Furthermore, the ability of BSO to inhibit the phosphoryla-

tion/degradation of InB-a and up-regulate cytokine produc-

tion demonstrated that this transcription factor could be

partially involved, but not exclusively required, in regulat-

Fig. 7. Schematic diagram of NF-nB activation circuits and oxygen-signaling mechanisms. GSSG reduction to GSH, which is blocked by BCNU, leads to

increasing intracellular stores of [GSSG], a potent inhibitor of NF-nB transcription factor DNA binding. The pathway leading to the formation of GSH by the

action of g-GCS is blocked by BSO, inducing an irreversible inhibition of NF-nB activation. ROS are key components of the pathways leading to the activation

of NF-nB, whose binding activity is obliterated by NAC and PDTC, potent scavengers of ROS. Although NAC is elevating [GSH], it is unknown whether this

mechanism induces NF-nB activation independently from the antioxidant effects of this inhibitor. PDTC elevates GSSG concentration by GSH oxidation, a

pro-oxidant effect characteristic of dithiocarbamates, thereby mediating NF-nB inhibition. Upon NF-nB DNA binding, cascades of hyperoxia-responsive genes

are activated, which have the potential to modulate cellular response to oxidative injury.

J.J. Haddad / Cellular Signalling 14 (2002) 879–897 885

ing oxidant-mediated pathways governing pro-inflammatory

cytokines. Although a NF-nB consensus binding site is

present in the promoter region of IL-1h, IL-6, IL-8 and

TNF-a genes, among other inflammatory targets, it cannot

be positively concluded whether these specific nB moieties

are indispensable for regulating cytokine expression and

release apart from coupling this mechanism to intracellular

redox-oxidant state, and whether other transcription factors

such as AP-1 and Oct-1 are likely to be involved. However,

there is apparently dissociation between ROS-dependent

and independent pathways governing the translocation/acti-

vation of NF-nB and that oxidative signaling tightly regu-

lates this pathway, along with the downstream pro-

inflammatory cytokine route. This is rather supported by

unequivocal evidence indicating a separation of oxidant-

initiated and redox-regulated mechanisms in the NF-nBsignal transduction pathway. Therefore, the InB-a/NF-nBpathway could be partially dissociated from that of redox-

dependent regulation of pro-inflammatory cytokines, in-

dicating that this transcription factor is not exclusively

ROS-sensitive and differentially implicated in regulating

pro-inflammatory cytokine signaling. This suggests the

involvement of a possible cross-talk among several pathways

working independently or in coherence to integrate ROS

signaling mechanisms governing the regulation of inflam-

mation. In support of this view, further studies showed that

the overall NF-nB signal transduction cascade begins with a

parallel series of stimuli-specific pathways through which

cytokines (such as TNF-a and IL-1h), oxidants (such as

H2O2 and mitomycin C), and phorbol ester (such as phorbol

12-myristate 13-acetate; PMA) individually and independ-

ently can initiate signaling. These initial pathways culminate

in a common pathway through which all of the stimulating

agents ultimately signal NF-nB activation [74]. The authors

distinguished the stimuli-specific pathways by showing that

the oxidative stimuli trigger NF-nB activation in only one of

two human T-cell lines (Wurzburg but not Jurkat), whereas

TNF-a and PMA readily stimulate in both lines. The

common pathway was proposed as the simplest way of

accounting for the common requirements and properties of

the signaling pathway. A redox-regulatory mechanism in this

common pathway was also included to account for the

previously demonstrated redox regulation of NF-nB activa-

tion in Jurkat cells (in which oxidants do not activate NF-

nB); the tyrosine phosphorylation cascade was subsequently

put in the common pathway by showing that kinase activity

(inhibited by herbimycin A and tyrphostin 47) is required for

NF-nB activation by all stimuli tested in both cell lines.

Since internal sites of oxidant production have been shown

to play a key role in the cytokine-stimulated activation of

NF-nB and since tyrosine kinase and phosphatase activities

are known to be altered by oxidants, these findings suggest

that intracellular redox status controls NF-nB activation by

regulating tyrosine phosphorylation event(s) within the

common step of the NF-nB signal transduction pathway

[2,46,74,159]. Another typical model is the development of a

stable aromatic acid decarboxylase expressing the Chinese

hamster ovary cell line to study the cytotoxic properties of

intracellular and extracellular dopamine and its effect on NF-

nB [120]. The relative impermeability of cells to dopamine,

but not to L-DOPA, allows the differentiation of extracellular

and intracellular dopamine cytotoxicity. In contrast to extrac-

ellular dopamine, intracellular dopamine toxicity was resist-

ant to antioxidant protection and did not require melanin

formation for its toxicity. Furthermore, it was demonstrated

that there was a rapid and potent activation of the stress-

inducible NF-nB by intracellular dopamine, which was also

largely insensitive to antioxidant inhibition. A distinctly

slower and less potent NF-nB activation by extracellular

dopamine was blocked by antioxidants and acetylsalicylic

acid, indicating the existence of a non-oxidative mechanism

of dopamine cytotoxicity [120].

3.3. Redox-mediated regulation of NF-jB

Cellular redox status regulates various aspects of cellular

functions such as proliferation, activation, growth inhibition

and cell death [6,12,13,48,57,60,69,88,166,178,194–198].

The term ‘oxidative stress’ indicates that the antioxidant

status of cells and tissues is altered by exposure to oxidants.

More and more evidence is accumulating that a proper

balance between oxidants and antioxidants is involved in

maintaining health and longevity, and altering this balance

may cause functional disorders and disease. Oxidative

stress plays a crucial role in a number of diseases includ-

ing neuro-degenerative diseases such as stroke, Parkinson’s

and Alzheimer’s diseases, cardiovascular and autoimmune

diseases, AIDS, as well as in ischemia reperfusion injuries.

Intracellular redox-regulating molecules and enzymes such

as glutathione, catalase, superoxide dismutase, glutathione

peroxidase and thioredoxin (TRX) maintain cellular redox

status. TRX and TRX reductase, for example, exist in all

living cells and constitute the major physiological

NADPH-dependent protein disulfide redox system. TRX

is a hydrogen donor for ribonucleotide reductase, the

essential enzyme for DNA synthesis, and has a large number

of other functions in redox regulation or defense against

oxidative stress. TRX (12 kDa) has successfully been

applied to reduction of disulfides of functional importance

in different proteins including fibrinogen, Factor X, Factor

VIII, insulin, a2-macroglobulin or IgG. TRX has been shown

to regulate the glucocorticoid receptor activity and a large

number of transcription factors like NF-nB or AP-1 [119].

Glutaredoxin (GRX), together with NADPH, glutathione

(GSH) and glutathione reductase (the glutaredoxin system),

is the other major protein disulfide oxido-reductase. Gluta-

redoxin 1 from Escherichia coli (9 kDa) or human cells (12

kDa) contains a redox-active disulfide and a binding site for

GSH. GRX is a hydrogen donor for ribonucleotide reductase;

it also operates as a general protein disulfide reductase with

specificity for GSH-mixed disulfides. Glutaredoxins have a

large number of functions overlapping with thioredoxins but

J.J. Haddad / Cellular Signalling 14 (2002) 879–897886

also unique functions related to redox regulation via gluta-

thione.

The role of redox signaling in modulating the pattern of

transcription and gene expression has been well documented

[1,2,24,52,54,162,175,199–201]. NF-nB dissociation by

kinase cascade is a primary step of NF-nB activation (Fig.

7) [112]. After dissociation from InB, NF-nB must go

through the redox regulatory pathway mediated by cellular

reducing catalysts, GSH and TRX. It is well established that

NF-nB cannot bind to the nB DNA sequence of the target

genes until it is reduced. Structural biological approaches

have shed new light on the redox regulation of NF-nB. First,in early 1995 two groups independently demonstrated the

three-dimensional structure of the NF-nB subunit p50

homodimer co-crystallized with the target DNA [150,158].

NF-nB appears to have a novel DNA-binding structure

called h-barrel, a group of h sheets stretching toward the

target DNA. There is a loop in the tip of the h-barrelstructure that intercalates with the nucleotide bases and is

considered to make a direct contact with the DNA. This

DNA-binding loop contains the cysteine 62 that acts as the

target of redox regulation by a proton donor, such as GSH

and TRX. Furthermore, another group recently solved the

3D structure of TRX molecule that is associated with the

DNA-binding loop of p50 by using NMR analysis

[68,90,172]. A boot-shaped hollow on the surface of TRX

containing the redox-active cysteines could stably recognize

the DNA-binding loop of p50 and is likely to reduce the

oxidized cysteine by donating protons in a structure-depend-

ent way. Therefore, the reduction of NF-nB is considered to

be specific [89,145,202].

NF-nB binding in vitro can be inhibited by agents that

modify free sulfhydryls [28,94,164]. For example, NF-nBbinding is eliminated after treatment with N-ethylmaleimide,

an alkylating agent, and diamide, an oxidizing agent. 2-

Mercaptoethanol can reverse the diamide effect and can act

synergistically with deoxycholate plus Nonidet P-40 in

converting inactive cytosolic NF-nB to an active DNA-

binding form [203]. It is, therefore, possible that modulation

of the redox state of NF-nB could represent a post-transla-

tional control mechanism for this factor. Furthermore, nBenhancer binding proteins isolated from the nuclei of

activated human T cells have been shown to produce two

distinct nucleoprotein complexes when incubated with the

nB element from the interleukin-2 receptor-a (IL-2Ra)

gene. These two DNA–protein complexes are composed

of at least four host proteins (p50, p55, p75, p85), each of

which shares structural similarity with the v-rel oncogene

product. Nuclear expression of these proteins is induced

with distinctly biphasic kinetics following phorbol ester

activation of T cells (p55/p75 early and p50/p85 late)

[171]. DNA–protein cross-linking studies have revealed

that the more rapidly migrating B2 complex contains both

p50 and p55 while the more slowly migrating B1 complex is

composed of p50, p55, p75, and p85. Site-directed muta-

genesis of the wild-type IL-2Ra nB enhancer (GGG-

GAATCTCCC) has revealed that the binding of p50 and

p55 (B2 complex) is particularly sensitive to alteration of

the 5Vtriplet of deoxyguanosine residues. In contrast, for-

mation of the B1 complex, reflecting the binding of p75 and

p85, critically depends upon the more 3V sequences of this

enhancer element. DNA binding by all four of these Rel-

related factors is blocked by selective chemical modification

of lysine and arginine residues, suggesting that both of

these basic amino acids are required for binding to the nBelement [171]. Similarly, covalent modification of free

sulfhydryl groups with diamide (reversible) or N-ethylma-

leimide (irreversible) results in a complete loss of DNA

binding activity. In contrast, mild oxidation with glucose

oxidase selectively inhibits p75 and p85 binding while not

blocking p50 and p55 interactions. These findings suggest

that reduced cysteine thiols play an important role in the

DNA binding activity of this family of Rel-related tran-

scription factors. A further role for redox regulation in

activation of NF-nB was suggested by the observation that

the DNA binding activity of free protein, but not preformed

DNA–protein complex, is inhibited by –SH modifying

agents but enhanced by reducing agents. Mutagenesis of

conserved cysteine residues in the p50 subunit identified

amino acid 62 as being important for DNA binding, as a

serine substitution at this position reduces DNA binding

affinity, but renders the protein insensitive to –SH modify-

ing agents [36]. DNA binding activity of the wild type

protein, but not the amino acid 62 mutant, was also

stimulated by thioredoxin while detection of disulphide

cross linked dimers in p50, but not the amino acid 62

mutant, suggests that thioredoxin stimulates DNA binding

by reduction of a disulphide bond involving cysteine 62.

The physiological relevance of these findings was supported

by the observation that co-transfection of a plasmid express-

ing human thioredoxin and an HIV LTR-driven reporter

construct resulted in an NF-nB-dependent increase in the

expression of the reporter gene [36,105,113,157,176]. Thus,

modification of p50 by thioredoxin, a gene induced by

stimulation of T-lymphocytes in parallel with NF-nB trans-

location, is a likely step in the cascade of events leading to

full NF-nB activation. In addition, the hypothesis that

cellular activation events occurring in T lymphocytes and

monocytes and mediated through translocation of the tran-

scription factor NF-nB are dependent upon the constitutive

redox status of these cells was investigated. Butylated

hydroxyanisole (BHA) was found to suppress not only

PMA- or TNF-induced, but also constitutive, HIV-enhancer

activity concomitant to an inhibition of NF-nB binding

activity in vitro [204]. This was also true for KBF (p50

homodimer) binding activity in U937 cells. Secretion of

TNF, the product of another NF-nB-dependent gene, wasabolished by BHA in PMA-stimulated U937 cells. The anti-

oxidative effect of BHAwas accompanied by an increase in

thiol, but not glutathione, content in stimulated and unsti-

mulated T cell, whereas TNF stimulation itself barely

modified the cellular thiol level. Furthermore, oxidative

J.J. Haddad / Cellular Signalling 14 (2002) 879–897 887

stress obtained by the addition of H2O2 could not by itself

induce NF-nB activation. These observations suggest that

TNF and PMA do not lead to NF-nB activation through

induction of changes in the cell redox status. Rather, TNF

and PMA can exert their effect only if cells are in an

appropriate redox status, because prior modification toward

reduction with BHA treatment prevents this activation. It

appears, therefore, that a basal redox equilibrium tending

toward oxidation is a prerequisite for full activation of

transduction pathways regulating the activity of NF-nB-dependent genes [92,204]. Furthermore, in macrophages,

NF-nB can be activated by H2O2 generated by the respira-

tory burst or added exogenously. The mechanism of H2O2

signaling may involve changes in the cellular redox state or

a redox reaction at the plasma membrane; however, the site

of H2O2 action cannot be readily ascertained because of its

membrane permeability. Ferricyanide, a non-permeable

redox active anion, activated NF-nB in the macrophage cell

line, J774A.1. In contrast with exogenous H2O2, activation

by ferricyanide did not correlate with net oxidation of

NAD(P)H or glutathione, suggesting that a trans-plasma

membrane redox reaction itself was the first signaling

process in NF-nB activation [8,155,169].

Phosphoseryl/threonyl protein phosphatase inhibitors,

okadaic acid and calyculin-A, failed to induce NF-nBnuclear translocation in several primary human cells

although a marked and rapid induction was observed in

their simian virus 40 transformed counterparts [170,205].

Inability to induce NF-nB cannot be due to a non-activat-

able system since NF-nB was strongly activated by TNF. It

is also unlikely that the differential induction was due to

differential sensitivity of primary and these inhibitors

equally inhibited transformed cells to phosphatase inhibitors

as the intracellular phosphatase activities of both cell types.

However, pretreatment with H2O2 or BSO, chemicals

known to elevate directly or indirectly the intracellular

free-radical levels, enabled okadaic acid to induce nuclear

translocation of NF-nB in primary cells. Conversely, cys-

teine, an antioxidant and precursor of the free radical

scavenger, glutathione, inhibited the induction of NF-nBby TNF in primary cells, and by okadaic acid or TNF in

transformed cells. These data, taken together, suggest that

free radical-dependent oxidation and protein phosphoryla-

tion are not independent modes of NF-nB induction, but are

both required for the release of NF-nB from InB. Further-more, the differential induction of NF-nB nuclear trans-

location by okadaic acid reflects intrinsic differences in the

intracellular oxidative/redox states. The induction of NF-nBby tumor necrosis factor in primary cells suggests that this

cytokine fulfills the requirement for oxidation, possibly by

inducing the production of free radicals [170,206]. In

support of this consensus, oxidative conditions have been

reported to potentiate the activation of NF-nB and AP-1 in

intact cells, but inhibit their DNA binding activity in vitro. It

has been shown that both the activation of NF-nB and the

inhibition of its DNA binding activity is modulated in intact

cells by the physiological oxidant glutathione disulphide

(GSSG). NF-nB activation in human T lineage cells (Molt-

4), for example, by 12-O-tetradecanoyl-phorbol 13-acetate

was inhibited by dithiothreitol and this was partly reversed

by the glutathione reductase inhibitor 1,3-bis(2-chlor-

oethyl)-1-nitrosourea (BCNU) or by H2O2, indicating that

GSSG may be required for NF-nB activation [184,207].

These effects of BCNU and H2O2 were not seen in

glutathione-depleted cells. However, NF-nB and AP-1 acti-

vation were potentiated by dithiothreitol if added to cell

cultures 1 h after the phorbol ester, indicating that a shift of

redox conditions may support optimal oxidative activation

with minimal inhibition of DNA binding. In addition, the

elevation of intracellular GSSG levels by BCNU before

stimulation suppressed the chloramphenicol acetyltransfer-

ase expression dependent on NF-nB but increased that

dependent on AP-1. This selective suppression of NF-nBwas also demonstrable by electrophoretic mobility shift

assays. In vitro, GSSG inhibited the DNA binding activity

of NF-nB more effectively than that of AP-1, while AP-1

was inhibited more effectively by oxidized thioredoxin

[50,93,100,102,146,184]. It was also demonstrated that

dihydrolipoate/a-lipoate redox couple, which is a cofactor

for mitochondrial dehydrogenases reactions, influences the

DNA binding activity of NF-nB. The elimination of dithio-

threitol in the electrophoretic mobility shift assay protocol

resulted in the inability to detect DNA binding activity of

activated NF-nB. The DNA binding activity was restored by

the addition of dihydrolipoate in the binding reaction

mixture. Inhibition of NF-nB DNA binding activity by in

vitro exposure to a sulfhydryl-oxidizing agent, diamide, was

also blocked by dihydrolipoate. In contrast, the addition of

the oxidized form, a-lipoate, inhibited the NF-nB DNA

binding activity. Coincidentally, pre-incubation of Jurkat

cells with dihydrolipoate potentiated and a-lipoate

inhibited, the okadaic acid-induced NF-nB activation as

detected by assessing its DNA binding activity [92,98,101,

102,136,149,208]. These results suggest that the redox

exchange exists between lipoate and NF-nB molecules.

Redox/ROS-mediated regulation of NF-nB is schematized

in Fig. 7.

In some unprecedented, but not uncommon, conditions,

NF-nB signaling pathways might exhibit redox-insensitive

liability. Renal tubular epithelial cells, for example, are

largely resistant to oxidant-induced injury despite their

capacity to accumulate relatively high concentrations of

potentially damaging prooxidant and thiol-depleting agents.

The hypothesis that such resistance may be attributable to a

lack or deficiency of signaling transduction pathways

through which reactive oxidants have been shown to pro-

mote the activation of NF-nB was tested. NF-nB was found

to be readily activated following exposure of cultured

normal rat kidney epithelial (NRK52E) cells to LPS. How-

ever, in contrast to findings with many other cell types, the

activation of NF-nB by LPS was not substantially altered

either by pretreatment of cells with the thiol antioxidant, N-

J.J. Haddad / Cellular Signalling 14 (2002) 879–897888

acetyl-L-cysteine (NAC), or by GSH depletion. Moreover,

reactive oxidants and oxidative stress-generating chemicals

were completely without effect with respect to NF-nBactivation in NRK52E cells, even following GSH depletion.

In contrast, LPS activation of NF-nB was substantially

attenuated by the intracellular Ca2 + chelator, Quin 2AM,

and by the Ca2 + -channel inhibitor, ruthenium red. More-

over, thapsigargin, a Ca2 + -ATPase inhibitor, promoted NF-

nB activation comparable to that observed by LPS. Addi-

tionally, staurosporine, a Ca2 + -dependent protein kinase C

inhibitor, substantially decreased LPS-mediated NF-nB acti-

vation [105,120,209]. These results demonstrate that the

LPS-inducible expression of NF-nB, in contrast to many

other cell types, is not responsive to oxidative stress and is

regulated, at least in part, by redox-insensitive modulation

of intracellular Ca2 + levels. Furthermore, heme–hemo-

pexin is used as a model for intravenous heme released in

trauma, stroke and ischemia–reperfusion to investigate the

role of transcription factors. A transient increase in cellular

protein oxidation occurs during receptor-mediated heme

transport from hemopexin, which is inhibited by the non-

permeable Cu(I) chelator, bathocuproinedisulfonate. Thus,

participation of surface redox process involving Cu(I) gen-

eration is proposed to be linked to the induction of the

protective proteins heme oxygenase-1 (HO-1) and metal-

lothionein-1 (MT-1) by heme–hemopexin. The region

(� 153 to � 42) in the proximal promoter of the mouse

MT-1 gene responds to heme– and CoPP–hemopexin in

transient transfection assays and contains metal-responsive

elements for MTF-1 and an antioxidant-responsive element

(ARE) overlapping a GC-rich E-box to which USF-1 and -2

bind. No decreases in DNA binding of the diamide-oxida-

tion sensitive USF-1 and USF-2 occur upon exposure of

cells to heme–hemopexin. MTF-1 and the ARE-binding

proteins are relatively resistant to diamide oxidation and are

induced approximately eight- and two-fold, respectively, by

heme–hemopexin. BCDS prevents the nuclear translocation

of MTF-1 and NF-nB by both heme– and CoPP–hemo-

pexin complexes, as well as MT-1 mRNA induction by

CoPP–hemopexin. Thus, copper is needed for the surface

oxidation events and yet the nuclear translocation of MTF-1

in response to hemopexin occurs via copper, probably

Cu(I)-dependent signaling cascades from the hemopexin

receptor rather than the oxidation per se. These findings

provide a basis for the highly tissue-specific expression and

function of NF-nB [70,104]. In addition, increasing evi-

dence indicates that intracellular redox-insensitive status

modulates the activity of various transcriptional factors,

including NF-nB [92,157,196,210]. Using primary pas-

sage-1 human tracheo-bronchial epithelial cell cultures and

an immortalized human bronchial epithelial cell line

(HBE1), it has been observed that TNF-a enhanced NF-

nB transcriptional activity (this observation was based on

gel mobility shift assays and IL-8 promoter–reporter gene

transfection studies). TNF-a activation coincided with the

translocation of RelA (p65) from the cytoplasm to the

nucleus. Furthermore, pretreatment with NAC or gluta-

thione inhibited TNF-a-induced activation of NF-nB tran-

scriptional activity and IL-8 promoter-mediated reporter

gene expression. In contrast, elevated TRX protein levels

in cells enhanced TNF-a-dependent NF-nB transcriptional

activity and IL-8 promoter activity. This observation was

independent of the manner in which TRX was elevated in

cells (e.g., by co-transfection with a FLAG-TRX expres-

sion clone, or by direct exposure to commercially avail-

able human TRX protein). Localization of TRX protein

by anti-TRX antibody indicated an accumulation of TRX

protein in the nucleus after TNF-a treatment. The nuclear

localization phenomenon was different from the major

cytosolic accumulation of glutathione and NAC [152].

This demonstrates that the movement of TRX into the

nucleus of airway epithelial cells occurs after an inflam-

matory stress, suggesting a compartmentalization effect of

thiol chemicals in the regulation of redox-dependent tran-

scriptional activity.

4. Oxygen radicals and redox regulation of HIF-1

signaling

The heterogeneous pO2 distribution in tissue ranging

from about 0 to 90 Torr at a constant arterial pO2 of about

100 Torr requires an oxygen-sensing system to optimize

specific organ functions. Cells located at the arterial inflow

have other metabolic properties or electrical activities than

cells located at the venous end. To meet the needs for such

different functions an oxygen sensor has to control short-

and long-term adaptation of cellular functions via regulation

of ion channel conductivity and gene expression. Oxygen is

the final acceptor of electrons in the synthesis of ATP by the

mitochondrial respiratory chain and is therefore an obliga-

tory substrate for energy transformations in most biological

systems. A reduction in the level of oxygen (hypoxia) in the

extracellular milieu severely limits the ability of cells to

perform energy-dependent functions and, if the hypoxia is

severe enough, it can lead to cell death. It is, therefore, not

surprising that elaborate mechanisms have evolved which

allow cells to detect changes in oxygen tension and protect

them against hypoxia. The long-range goal of biomedical

research is to identify the molecular and cellular mecha-

nisms by which cells detect changes in oxygen tension and

how this signal is transduced into the nuclear events

responsible for altered gene expression during hypoxia.

Altered gene expression is essential for development of a

hypoxia tolerant phenotype, which is more resistant to cell

damage or death. Gene regulation is a complex biological

process that results from molecular interactions among

nuclear protein factors (transcription factors) and DNA

control sequences. These protein–DNA interactions often

occur as the result of an extracellular stimulus that is

transmitted to the nucleus by a specific signal transduction

pathway(s). Although numerous stimuli have been identi-

J.J. Haddad / Cellular Signalling 14 (2002) 879–897 889

fied which regulate gene expression, perhaps none is more

intriguing than reduced oxygen (hypoxia). Hypoxia-induced

gene expression has been implicated in a number of

physiological processes, including erythropoiesis, carotid

body chemoreceptor function, and angiogenesis, all of

which enhance the delivery of oxygen to tissue [168].

Genes involved in mediating each of these important

processes are normally activated by long-term (hours to

days) rather than acute (seconds to minutes) episodes of

hypoxia [3,211].

4.1. Oxidative stress-mediated regulation of HIF-1

An important transcription factor that is a crucial

regulatory element in sensing hypoxic conditions and

integrating an adapted response via gene expression of

oxygen-sensitive enzymes and cofactors is the HIF-1 (Fig.

8) [3,180]. The signal transduction components which link

the availability of oxygen to the activation of these tran-

scription factors are poorly defined, but are broadly

believed to hinge on the free abundance of oxidants (i.e.,

ROS) in the cytosol. In the case of HIF-1a, for example,

post-translational stability, nuclear translocation by the aryl

hydrocarbon receptor translocator (ARNT) and consensus

DNA binding are coupled with oxygen-associated changes

in both conformation and activity of a ferroheme contain-

ing protein, believed to express peroxide generation via a

NADPH oxidase-type activity [3,45]. Hypoxic cessation of

peroxide production, for example, mediates HIF-1a stabi-

lization, nuclear translocation and gene expression. The

myriad of genes directly targeted by HIF-1a is given in

Table 2.

The stability and activity of HIF-1a, first identified as a

DNA-binding activity expressed under hypoxic conditions,

increase exponentially when pO2 is lowered. The ubiquitous

activation of HIF-1a is thus consistent with the significant

role that this transcription factor plays in coordinating

adaptive responses to hypoxia [3,45]. Low oxygen condi-

tions are known to induce alterations in gene expression

serving for the adaptation to the environmental conditions.

HIF-1 consists of two subunits: HIF-1a, which is unique to

the oxygen-response, and HIF-1h, previously known as

ARNT. Whereas ARNT is constitutively expressed, hypoxia

protects HIF-1a from proteolytic degradation in protea-

somes by so far unknown mechanisms. HIF-1, therefore,

is a heterodimeric complex consisting of a- and h-subunits.ARNT (HIF-1h) can dimerize with the aryl hydrocarbon

receptor as well as with HIF-1a, because both contain

Fig. 8. Potential oxygen sensing mechanisms and the role of the transcription factor HIF-1.

Table 2

Direct HIF-1 target genes

Glucose/Energy metabolism and cell proliferation/viability

Adenylate Kinase 3, Aldolase A, Aldolase C, Enolase 1 (ENO-1), Glucose

Transporter 1, Glucose Transporter 3, Glyceraldehyde-3-phosphate

Dehydrogenase, Hexokinase 1, Hexokinase 2, Insulin-like Growth

Factor 2 (IGF-2), IGF Binding Protein 1 (IGFBP-1), IGFBP-3, Lactate

Dehydrogenase A, Phosphoglycerate Kinase 1, Pyruvate Kinase M, p21,

Transforming Growth Factor

Erythropoiesis and iron metabolism

Ceruloplasmin, Erythropoietin (EPO), Transferrin, Transferrin Receptor

Vascular development/remodeling and vasomotor tone

Adrenergic Receptor, Adrenomedullin, Endothelin-1, Heme Oxygenase 1

(HO-1), Nitric Oxide Synthase 2, Plasminogen Activator Inhibitor 1,

Vascular Endothelial Growth Factor (VEGF), VEGF Receptor FLT-1

J.J. Haddad / Cellular Signalling 14 (2002) 879–897890

helix– loop–helix and PAS (‘Per-ARNT-Sim’) domains.

Whereas HIF-1h is constitutively expressed under normoxic

conditions, HIF-1a is rapidly degraded by the ubiquitin–

proteasome system. However, under hypoxic conditions,

HIF-1a protein stabilizes and accumulates, thus allowing

the heterodimer to translocate to the nucleus and to bind

specific promoter moieties of selective genes encoding

erythropoietin (EPO), vascular endothelial growth factor

(VEGF), glycolytic enzymes and glucose transporters

(GLUT), as well as cytokines and other inflammatory

mediators [3,45]. Adaptive responses to hypoxia, therefore,

involve the regulation of gene expression by HIF-1a, at

least in part, whose expression, stability and transcriptional

activity are reported to increase exponentially on lowering

pO2. During hypoxia, multiple systemic responses are

induced, including angiogenesis, erythropoiesis and glycol-

ysis. HIF-1a is a crucial mediator for increasing the effi-

ciency of O2 delivery through EPO and VEGF [108]. A

well-controlled process of adaptation parallels this mecha-

nism with decreased O2 availability through expression and

activation of glucose transporters and glycolytic enzymes.

EPO, for example, is responsible for increasing blood O2-

carrying capacity by stimulating erythropoiesis; VEGF is a

transcriptional regulator of vascularization and glycolytic

transporters and enzymes increase the efficiency of anaero-

bic generation of ATP, the vital biological currency. It is

expected that any reduction of tissue oxygenation in vivo

and in vitro would, therefore, provide a mechanistic stim-

ulus for a graded and adaptive response mediated by HIF-

1a. HIF-1a, therefore, may play an important role not only

in regulating the transcription of pO2-controlled genes and

energy homeostasis, but also in influencing immune

responses. However, the mechanism of cytokine-dependent

regulation of the translocation and activation of HIF-1a is

currently unknown and primitive. Cytokines act as major

participants in mediating molecular responses in physiology

and pathophysiology. There is accumulating evidence, more-

over, suggesting that the conventionally known ‘pro-inflam-

matory’ cytokines act as O2-sensitive mediators, indicating

the potential to integrate O2-linked pathways mediated by

cytokines via a ROS-dependent mechanism. Direct contact

of the respiratory epithelium, for instance, with dynamic

fluctuations in pO2 at the alveolar space blood barrier

junction during pulmonary-based ventilation is likely, there-

fore, to up-regulate the transcription, biosynthesis and mobi-

lization of cytokines. This is strengthened by the evidence

implicating the epithelium in a front-line defense strategy

activated in preparation for birth into an O2-rich environ-

ment. Furthermore, the generation of ROS might induce

pulmonary damage; however, moderate oxidative stress can

induce a signaling mechanism mediated, at least in part, by

O2-responsive cytokines. ROS, for instance, can induce pro-

inflammatory cytokine biosynthesis and this response can be

abrogated by selective antioxidants, suggesting an integral

role of endogenous ROS. As such, cytokines could form a

pivotal link in ROS-dependent pathways leading to the

activation of redox-sensitive transcription factors such as

HIF-1a, whose up-regulation determines the specificity of

cellular responses to oxidative stress.

Recent investigations have revealed a novel role for ROS

signaling in mediating a non-hypoxic effect of cytokines on

HIF-1a stabilization, nuclear translocation and activation

during normoxia. Despite the fact that HIF-1a was recog-

nized as a transcriptional activator prevailing under hypoxic

conditions, ROS signaling pathways that mediate the regu-

lation of HIF-1a have emerged only recently. Consistent

with this notion, it was reported that a non-hypoxic pathway

mediating the effect of cytokines in regulating the stabiliza-

tion, translocation and activation of HIF-1a in a ROS-

sensitive mechanism. These results suggest that hypoxia

may not be the only major player in HIF-1a regulation and

that this pathway mediated by inflammatory cytokines may

play a major role in controlling HIF-1a regulation in a non-

hypoxic environment [3,45,85,129,211]. The concept, there-

fore, has been put forward that ROS and phosphorylation/

dephosphorylation events are master regulators of HIF-1a

induction and activation; however, the underlying pathways

and potential signaling mediators likely to be implicated

have yet to be identified. Regarding the mechanism of non-

hypoxic, ROS-dependent regulation of HIF-1a, a major role

for mitochondrial ROS generated at complex III site was

reported, thereby causing the accumulation of HIF-1a pro-

tein ostensibly responsible for the initiation of gene expres-

sion. Furthermore, depletion of the mitochondrial genome

has been shown to reverse ROS-mediated induction of HIF-

1a. In addition, it has been reported that HIF-1a-dependent

transcriptional activity in the induction of VEGF expression

has defined a novel hypoxia-independent mechanism regu-

lating vascular remodeling. Further afield, recent evidence

suggested that the RNS pathway regulates the stability and

activity of HIF-1a. For instance, it was shown that the

expression of NO synthase could cause HIF-1a accumula-

tion, thus underscoring the role of NO as an intracellular

activator of this transcription factor (Fig. 9) [221].

Fig. 9. The regulation of HIF-1a in oxidative stress, particularly involving a

crucial role for NO is observed.

J.J. Haddad / Cellular Signalling 14 (2002) 879–897 891

The molecular pathways defined by cytokines that poten-

tially mediate HIF-1a regulation, are not well understood.

Recently, for instance, a novel role for IL-1h and TNF-a as

regulatory mediators in stimulating the DNA-binding activ-

ity of HIF-1a has been reported, indicative of the conclusion

that this transcription factor is likely to be involved in

modulating gene expression during inflammation [85,129].

The augmented activity of HIF-1a due to IL-1h was sub-

sequently attributed, at least in part, to increased HIF-1a

protein abundance under non-hypoxic conditions, whereas

the effect of TNF-awas thought to be due to the concomitant

activation of certain proteins that were proposed to be part of

the activated complex. Moreover, cAMP-responsive element

binding protein (CREB) has been implicated in augmenting

HIF-1a activity within its site in the EPO gene enhancer.

Although no effect of cytokines on cAMP levels and protein

kinase A (PKA) activity was presented, it has been reported

that cAMP analogues prevented the in vitro suppression of

EPO production mediated by cytokines [3,45,222]. Amongst

pro-inflammatory cytokines, TNF-a and IL-1h have perhaps

been most exhaustively investigated. For example, IL-1h,like TNF-a, exerts its pleiotropic activities through ligand-

induced cross-linking of specific receptors [167]. During

inflammation, cytokines, including TNF-a and IL-1h, tran-siently activate neutrophils and macrophages, thereby caus-

ing enhanced production of O2� possibly via the activation of

NADPH oxidase. This oxidative burst involves a rapid but

transient release of ROS as a crucial part of the defense

mechanisms against invading microbial pathogens and tumor

cell metastasis. Although TNF-a and IL-1h are primarily

produced by macrophages, accumulating evidence suggests

that other cell types such as endothelial and epithelial cells

release IL-1h, IL-6, TNF-a and other inflammatory media-

tors, thereby amplifying the inflammatory responses by

activating and recruiting inflammatory cells. In this respect,

it has been reported that cytokines up-regulate intracellular

accumulation of ROS. More recently, well-defined studies

indicated that cytokines increase mitochondrial ROS gener-

ation, suggesting that their effect could be mediated through a

ROS-sensitive mechanism [3,45]. The results reported by

Haddad and Land [85,129] indicate a potential role for

cytokines in inducing the accumulation of intracellular

ROS, thereby reinforcing the notion that these mediators

exert their effect on transcription factors including HIF-1a

through a ROS-dependent mechanism. Regarding the likely

source of ROS production engendered by cytokines, at least

two sources may be involved: (i) the membrane-bound

NADPH oxidase and (ii) the mitochondrial respiratory chain.

Although ROS, in general, may be involved in mediating the

effect of cytokines on HIF-1a induction, the latter mecha-

nism probably predominates because blockade of mitochon-

drial respiration abrogates cytokine-dependent activation of

HIF-1a. Furthermore, evidence is provided that different

ROS species (O2� , OH and H2O2) may mediate the effect of

cytokines on HIF-1a stabilization, localization and acti-

vation. The observation that a non-hypoxic pathway medi-

ates the effect on HIF-1a which involves ROS generated

within the mitochondrial complex is supported by unequiv-

ocal evidence since diphenylene iodonium (DPI), an inhib-

itor of complex I nicotinamide adenine dinucleotide

phosphate-dependent oxidase (which blockades the conver-

sion of ubiquinone! ubiquinol), abrogates cytokine-medi-

ated activation of HIF-1a, indicating a crucial role for

mitochondria-derived ROS in HIF-1a signaling. Although

it has been suggested that ROS mediate their signaling by

affecting kinases and/or phosphatase activities, the precise

downstream pathway(s) affected by ROS, which govern HIF-

1a translocation/activation, have yet to be identified. It is

concluded, therefore, that the production of ROS is clearly

involved in cytokine-mediated normoxic regulation of HIF-

1a stabilization, translocation and activation [42,221].

In support of the aforementioned observations, Nickel

(Ni2 + ) and cobalt (Co2 + ) were reported to mimic hypoxia

and were used as a tool to study the role of oxygen sensing

and signaling cascades in the regulation of hypoxia-indu-

cible gene expression [212]. These metals can produce

oxidative stress; therefore, it was conceivable that ROS

may trigger signaling pathways resulting in the activation

of HIF-1 transcription factor and up-regulation of hypoxia-

related genes. It was found that the exposure of A549 cells

to Co2 + or Ni2 + produced oxidative stress, and although

Co2 + was a more potent producer of ROS than Ni2 + , both

metals equally increased the expression of Cap43, a hypo-

xia-regulated gene [116]. The co-administration of H2O2

with metals induced more ROS; however, this did not

further increase the expression of Cap43 mRNA. The free

radical scavenger 2-mercaptoethanol completely suppressed

ROS generation by CoCl2 and NiCl2 but did not diminish

the induced Cap43 gene expression. Ni2 + , hypoxia and

desferrioxamine as assessed in transient transfection assays,

stimulated the activity of the HIF-1 transcription factor, but

this activation was not diminished when oxidative stress

was attenuated nor was HIF-dependent transcription

enhanced by H2O2. It is concluded that ROS are produced

during the exposure of cells to metals that mimic hypoxia,

but the formation of ROS was not involved in the activation

of HIF-1-dependent genes (Fig. 10).

On the hypoxic mechanisms that regulate HIF-1, cad-

mium (Cd) has been reported as a substantial industrial and

environmental pollutant that seriously impairs erythropoi-

esis. Cd has been demonstrated to aggravate anemia by

suppressing erythropoietin gene expression in anemic

patients. As hypoxic induction of erythropoietin mRNA

depends on HIF-1, it was hypothesized that Cd suppresses

the hypoxic activation of HIF-1. In hypoxic Hep3B cells,

Cd suppressed all mRNAs of various genes, which are

known to be upregulated by HIF-1 activation under hypo-

xia, in a dose-dependent manner [213]. Cd inhibited the

hypoxia-induced activity of luciferase in 293 cells, which

were transfected with a reporter plasmid carrying a hypoxia

response element. By electrophoretic mobility gel shift

assay, Cd inhibited the DNA-binding activity of HIF-1 in

J.J. Haddad / Cellular Signalling 14 (2002) 879–897892

hypoxic Hep3B cells. Cd reduced the amount of HIF-1a

protein in hypoxia, whereas it did not affect HIF-1a mRNA

levels. Moreover, Cd inhibited HIF-1a accumulation in-

duced by cobalt and desferrioxamine. Antioxidants and a

proteasome inhibitor prevented the HIF-1a degradation

caused by Cd. The possibility that oxidative stress mediates

this action of Cd was subsequently examined. Cd did not

affect protein oxidation and reduced glutathione levels in

hypoxic cells. These results indicate that Cd triggers a redox/

proteasome-dependent degradation of HIF-1a protein,

reducing HIF-1 activity and in turn suppressing the hypoxic

induction of hypoxia-inducible genes [213]. Furthermore,

iron chelators are pluripotent neuronal anti-apoptotic agents

that have been shown to enhance metabolic recovery in

cerebral ischemia models [127]. The precise mechanism(s)

by which these agents exert their effects remains unclear.

Recent studies have demonstrated that iron chelators activate

a hypoxia signal transduction pathway in non-neuronal cells

that culminates in the stabilization of HIF-1 and increased

expression of gene products that mediate hypoxic adaptation.

Zaman et al. [127] examined the hypothesis that iron

chelators prevent oxidative stress-induced death in cortical

neuronal cultures by inducing expression of HIF-1 and its

target genes. It was reported that the structurally distinct iron

chelators desferrioxamine mesylate and mimosine prevent

apoptosis induced by glutathione depletion and oxidative

stress in embryonic cortical neuronal cultures. The protec-

tive effects of iron chelators are correlated with their ability

to enhance DNA binding of HIF-1 and activating tran-

scription factor 1 (ATF-1)/CREB to the hypoxia response

element in cortical cultures and the H19-7 hippocampal

neuronal cell line. It was shown that mRNA, protein, and/or

activity levels for genes whose expression is known to be

regulated by HIF-1, including glycolytic enzymes, p21

(waf1/cip1), and erythropoietin, are increased in cortical

neuronal cultures in response to iron chelator treatment.

Finally, it was demonstrated that cobalt chloride, which also

activates HIF-1 and ATF-1/CREB in cortical cultures, also

prevents oxidative stress-induced death in these cells. Alto-

gether, these results suggest that iron chelators exert their

neuroprotective effects, in part, by activating a signal trans-

duction pathway leading to increased expression of genes

known to compensate for hypoxic or oxidative stress. In

contrast, one hypothesis for oxygen sensing has postulated

that ROS generated at mitochondrial complex III are the

initiators of the hypoxic signal. It was found that mitochon-

drial DNA-devoid (Uj) cells have a normal response to

hypoxia, measured at the level of HIF-1a protein stabiliza-

tion, nuclear translocation, and its transcriptional activation

activity. Furthermore, overexpression of catalase, either in

the mitochondria or in the cytosol, fails to modify the

hypoxia response indicating that H2O2 is not a signaling

molecule in the hypoxic signaling cascade that culminates

with HIF-1 activation [4,214,215,222].

Fig. 10. Oxygen sensing proposed mechanisms for the regulation of gene transcription and the involvement of HIF-1 as a hypoxia-mediated transcriptional

activity (see discussion for further details). Abbreviations: AA, arachidonic acid; ARNT, aryl receptor hydrocarbon nuclear translocator; CREB, cAMP-

responsive element binding protein; CBP, CREB-binding protein; DAG, diacyl glycerol; ECF, extracellular fluid; HIF-1, hypoxia-inducible factor-1; ICF,

intracellular fluid; IP3, inositol triphosphate; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; ROS, reactive oxygen species; SAPK, stress-

activated protein kinase.

J.J. Haddad / Cellular Signalling 14 (2002) 879–897 893

4.2. Redox-mediated regulation of HIF-1

Cellular redox state is closely related to hypoxia by

changes in the expression of redox-regulated genes and

the generation of ROS (Fig. 10) [216,221,222]. Direct

evidence was provided indicating that alteration of cellular

redox states by treating cells with H2O2 or dithiothreitol

impairs hypoxia signaling mechanisms and the expression

of HIF-1a protein in hypoxic cells. In addition, HIF-1

DNA-binding activity in vitro is sensitive to oxidizing

reagents diamide and H2O2 and the alkylating agent N-

ethylmaleimide. Of note, the activity of N-ethylmaleimide-

inactivated HIF-1 can be partially restored by addition of

nuclear extract from non-hypoxic cells [53]. Furthermore,

the regulation of HIF-1 activity is primarily determined by

the stability of the HIF-1a protein. Both HIF-1a and HIF-

1h mRNAs were constitutively expressed in HeLa and

Hep3B cells with no significant induction by hypoxia

[217]. However, the HIF-1a protein was barely detectable

in normoxic cells, even when HIF-1a was over-expressed,

but was highly induced in hypoxic cells, whereas HIF-1hprotein levels remained constant, regardless of pO2. Hypo-

xia-induced HIF-1 DNA-binding as well as the HIF-1a

protein were rapidly and drastically decreased in vivo

following an abrupt increase to normal oxygen tension.

Moreover, short pre-exposure of cells to H2O2 selectively

prevented hypoxia-induced HIF-1 binding via blocking

accumulation of HIF-1a protein, whereas treatment of

hypoxic cell extracts with H2O2 had no effect on HIF-1

binding [217,222]. These observations suggest that an intact

redox-dependent signaling pathway is required for destabi-

lization of the HIF-1a protein. In hypoxic cell extracts, HIF-

1 DNA binding was reversibly abolished by sulfhydryl

oxidation. Furthermore, the addition of reduced thioredoxin

to cell extracts enhanced HIF-1 DNA binding. Consistent

with these results, overexpression of thioredoxin and Ref-1

significantly potentiated hypoxia-induced expression of a

reporter construct containing the wild-type HIF-1 binding

site, indicating that the activation of HIF-1 involves redox-

dependent stabilization of HIF-1a protein [80,218,221,222].

HIF-a protein is continuously synthesized, but is rapidly

degraded by the ubiquitin–proteasome system under oxic

conditions. Hypoxia, transition metals, iron chelators and

several antioxidants stabilize the HIF-a proteins, allowing

the formation of the transcriptionally active HIF complex.

However, the sequences and mechanisms involved in the

regulated degradation of the a protein subunits are poorly

understood. Analysis of the available cloned sequences of

human and mouse members of the HIF-a family of proteins

revealed an area of about 15 amino acids with strong

sequence conservation between all the members [219–

221]. This area corresponds to the region encompassing

amino acids 557–571 of the hHIF-1a subunit. Fragments of

HIF-1a and HIF-3a proteins containing this conserved

sequence were able to confer hypoxia regulation when

expressed as fusion proteins in Hep3B cells. Regulation

was observed with all the known hypoxia ‘‘mimics,’’ includ-

ing the reducing thiol donor N-mercaptopropionylglycine.

Furthermore, selective alanine substitutions of amino acids

561–568 stabilized the protein in normoxic conditions. In

addition, transfection with an expression vector containing a

fragment of hHIF-1a comprising amino acids 540–580

enhanced transactivation activity of the full-length hHIF-

1a protein. These results suggest that the above-mentioned

conserved sequences are likely to be involved in the hypoxic

stabilization of HIF-a proteins. The mechanisms and the

interacting ubiquitin-ligases involved in the selective degra-

dation process, however, remain unknown. HIF-1a and HLF

are two highly related basic helix–loop–helix/Per-ARNT-

Sim (bHLH/PAS) homology transcription factors. Despite

strong similarities in their activation mechanisms (e.g., they

both undergo rapid hypoxia-induced protein stabilization,

bind identical target DNA sequences, and induce synthetic

reporter genes to similar degrees), they are both essential for

embryo survival via distinct functions during vascularization

(HIF-1a) or catecholamine production (HLF). It is currently

unknown how such specificity of action is achieved. In this

respect, it was reported here that DNA binding by HLF, but

not by HIF-1a, is dependent upon reducing redox condi-

tions. In vitro DNA binding and mammalian two-hybrid

assays showed that a unique cysteine in the DNA-binding

basic region of HLF is a target for the reducing activity of

redox factor Ref-1 [220–222]. Although the N-terminal

DNA-binding domain of HIF-1a can function in the absence

of Ref-1, it was found that the C-terminal region containing

the transactivation domain requires Ref-1 for full activity.

These data reveal that the hypoxia-inducible factors are

subject to complex redox control mechanisms that can target

discrete regions of the proteins, thereby establishing a dis-

criminating control mechanism for differential regulation of

HIF-1a and HLF activity.

5. Conclusion and future prospects

The study of gene expression and gene regulation is

critical in the development of novel gene therapies. Reac-

tive oxygen and nitrogen species (oxidative stress) are

produced in health and disease. The antioxidant defense

system—a complex system that includes intracellular

enzymes, non-enzymatic scavengers, and dietary compo-

nents—normally controls the production of ROS. Oxida-

tive stress occurs when there is a marked imbalance

between the production and removal of reactive oxygen

and nitrogen species. This imbalance arises when antiox-

idant defenses are depleted or free radicals are overpro-

duced. A growing body of evidence also exists showing

that enhancement of the oxidative stress antioxidant

defense system can reduce markers of oxidative stress.

Recognition of reactive species and redox-mediated protein

modifications as potential signals may open up a new field

of cell regulation via specific and targeted genetic control

J.J. Haddad / Cellular Signalling 14 (2002) 879–897894

of transcription factors and thus can provide us with a

novel way of controlling disease processes.

Acknowledgements

The author’s own publications are financially supported

by the Anonymous Trust (Scotland), the National Institute

for Biological Standards and Control (England), the

National Institutes of Health (NIH; USA), the Tenovus

Trust (Scotland), the UK Medical Research Council (MRC,

London) and the Wellcome Trust (London). Dr. John J.

Haddad holds the George John Livanos prize (London) and

the NIH (California, USA) award fellowship.

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