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International Immunopharmacology 4 (2004) 1249–1285
Review article
Hypoxia and the regulation of mitogen-activated protein kinases:
gene transcription and the assessment of potential
pharmacologic therapeutic interventions
John J. Haddad*
Severinghaus-Radiometer Research Laboratories, University of California, San Francisco, CA, USA
Received 15 May 2004; received in revised form 9 June 2004; accepted 15 June 2004
Abstract
Oxygen is an environmental/developmental signal that regulates cellular energetics, growth, and differentiation processes.
Despite its central role in nearly all higher life processes, the molecular mechanisms for sensing oxygen levels and the pathways
involved in transducing this information are still being elucidated. Altering gene expression is the most fundamental and
effective way for a cell to respond to extracellular signals and/or changes in its microenvironment. During development, the
expression of specific sets of genes is regulated spatially (by position/morphogenetic gradients) and temporally, presumably via
the sensing of molecular oxygen available within the microenvironment. Regulation of signaling responses is governed by
transcription factors that bind to control regions (consensus sequences) of target genes and alter their expression in response to
specific signals. Complex signal transduction during hypoxia (deficiency of oxygen in inspired gases or in arterial blood and/or
in tissues) involves the coupling of ligand–receptor interactions to many intracellular events. These events basically include
phosphorylations by tyrosine kinases and/or serine/threonine kinases, such as those of mitogen-activated protein kinases
(MAPKs), a superfamily of kinases responsive to stress nonhomeostatic conditions. Protein phosphorylations imposed during
hypoxia change enzyme activities and protein conformations, and the eventual outcome is rather complex, comprising of an
1567-5769/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.intimp.2004.06.006
Abbreviations: NAC, N-acetyl-L-cysteine; AP, activating protein; ATF, activating transcription factor; bFGF, basic fibroblast growth factor;
CaM, calmodulin; PKM, Ca2+/CaM-dependent protein kinase; CT, cardiotrophin; COXI, cytochrome oxidase I; DTT, dithiothreitol; dHIF,
Drosophila HIF; Draf, Drosophila Raf; EGF, epidermal growth factor; ERT kinase, EGF receptor threonine kinase; EPO, erythropoietin; ECM,
extracellular matrix; ERK, extracellular signal-regulated kinase; GPCR, G-protein-coupled receptor; GSH, glutathione; hep, hemipterous; HGF,
hepatocyte growth factor; HHV, human herpes virus; HMEC, human microvascular endothelial cells; HIF, hypoxia-inducible factor; HPTF, HIF
proteasome-targeting factor; HRE, hypoxia response element; InB, inhibitory nB; IFN, interferon; IL, interleukin; JNK, Jun N-terminal kinase;
KSHV, Kaposi’s sarcoma-associated herpes virus; LPS, lipopolysaccharide endotoxin; NMDA, N-methyl-D-aspartate; MAP-2 kinase,
microtubule-associated protein-2 kinase; MAPK, mitogen-activated protein kinase; MKP, MAPK phosphatase; MKK, MAPK kinase; MEKK,
MKK kinase; MAPKAP-K, mitogen-activated protein kinase-activated protein kinase; MBP kinase, myelin basic protein kinase; NGF, nerve
growth factor; NO, nitric oxide; NF-nB, nuclear factor-nB; NIK, NF-nB-inducing kinase; NLS, nuclear localization signal; OA, okadaic acid;
Ppase-1, phosphatase-1; PDGF, platelet-derived growth factor; PC, preconditioning; PKA, protein kinase A; PKC, protein kinase C; PTK,
protein tyrosine kinase; puc, puckered; RTK, receptor tyrosine kinase; RHD, Rel homology domain; rho, rhomboid; RSK, ribosomal S6 protein
kinase; SRF, serum response factor; spi, Spitz; SAPK, stress-activated protein kinase; Sp, substance P; SOD, superoxide dismutase; Tor, torso;
TGF, transforming growth factor; TNF, tumor necrosis factor; TyrK, tyrosine kinase; UB, ubiquitin; VEGF, vascular endothelial growth factor;
vn, vein; pVHL, von Hippel–Lindau protein.
* C/o Prof. Bared Safieh-Garabedian, Department of Biology, American University of Beirut, Bliss St., Beirut, 110236, Lebanon. Tel.:
+961-1-350-000; fax: +961-1-374-374.
E-mail address: johnjhaddad@yahoo.co.uk (J.J. Haddad).
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–12851250
alteration in cellular activity and changes in the programming of genes expressed within the responding cells. These molecular
changes serve as signals that are crucial for cell survival under contingent conditions imposed during hypoxia. This review
correlates current concepts of hypoxic sensing pathways with hypoxia-related phosphorylation mechanisms mediated by
MAPKs via the genetic and pharmacologic regulation/manipulation of specific transcription factors and related cofactors.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Cell signaling; Development; Disease; Gene regulation; Hypoxia; Kinase; MAPK; Transcription factors
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1250
2. MAPK signaling modules and pathways: a network recap and current concepts . . . . . . . . . . . . . . . . . 1251
2.1. MAPK signaling pathways as viewed through their identification and bifurcations. . . . . . . . . . . . 1251
2.2. MAPK signaling as viewed through receptor and nonreceptor coupled cofactors . . . . . . . . . . . . . 1253
2.3. MAPK signaling and redox-mediated regulation of kinases/phosphatases. . . . . . . . . . . . . . . . . 1257
3. Hypoxia-mediated regulation of MAPK signaling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . 1258
3.1. Hypoxia and oxygen-sensing mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1258
3.2. Hypoxia and MAPK signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1261
4. Hypoxia-mediated regulation of transcription factors and gene transcription: the role of
MAPK-related signaling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1263
4.1. MAPK-mediated regulation of HIF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1263
4.1.1. HIF and oxygen sensing—an overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1263
4.1.2. HIF and MAPK regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1266
4.2. MAPK-mediated regulation of nuclear factor-nB (NF-nB) . . . . . . . . . . . . . . . . . . . . . . . . 1268
4.3. MAPK-mediated regulation of activating protein-1 (AP-1) . . . . . . . . . . . . . . . . . . . . . . . . 1272
5. The role of MAPK signaling pathways in hypoxia- or anoxia-tolerant organisms . . . . . . . . . . . . . . . . 1273
6. Summary, conclusion, and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1276
Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1276
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1277
1. Introduction Oxygen is a unique stimulus that readily diffuses
Gene regulation (activation/repression) is a com-
plex biological process that results from molecular
interactions among nuclear protein factors (transcrip-
tion factors) and DNA consensus control sequences
[1–5]. These protein–DNA interactions often occur as
a result of an extracellular stimulus that is transmitted
to the nucleus by a specific signal transduction path-
way(s). Although numerous stimuli that regulate gene
expression have been identified, perhaps none is more
intriguing than reduced oxygen (hypoxia) [2,6–10].
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 tissues [1,9,10].
throughout the cell; thus, hypoxia may regulate gene
expression by a variety of different mechanisms in
different cell types. Major challenges, however, in-
clude: (i) identification of oxygen sensory mecha-
nisms; (ii) further identification and characterization
of oxygen-regulated signal transduction pathways; (iii)
identification of additional genes that are regulated by
hypoxia; and (iv) understanding the role that these
genes play in regulating the response to hypoxia. Such
information will provide new insights into hypoxia-
regulated physiological (signaling and developmental)
and pathological (disease) processes [1–4,6–10]. In
this review, I particularly stress emphasis on the
current understanding of the role of hypoxia in regu-
lating the mitogen-activated protein kinase (MAPK)
pathways via upstream and downstream mechanisms.
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–1285 1251
2. MAPK signaling modules and pathways: a
network recap and current concepts
2.1. MAPK signaling pathways as viewed through
their identification and bifurcations
Signal transduction at the cellular level refers to the
movement of signals from outside (extracellular) the
cell to inside (intracellular) the cell [11–18]. The
movement of signals can be simple, like that associa-
ted with receptor molecules of the acetylcholine class
receptors that constitute channels, which, upon ligand
interaction, allow signals to be passed in the form of
small ion movement, either into or out of the cell [11–
13,18]. These ion movements result in changes in the
electrical potential of the cells that, in turn, propagates
the signal spatially along the cell. More complex
signal transduction, furthermore, involves the coup-
ling of ligand–receptor interactions to many intracel-
lular events. These events include phosphorylations
by tyrosine kinases (TyrKs) and/or serine/threonine
kinases [11–15,17,18]. Protein phosphorylation
changes enzyme activities and protein conformations.
The eventual outcome is an alteration in cellular
Fig. 1. A model of gene regulation where the switch on/off mediates a ser
and phosphatases, respectively. This sequential propagation of signals occ
activity and changes in the programming of genes
expressed within the responding cells. Phosphoryla-
tion and dephosphorylation mechanisms in the regu-
lation of transcription are depicted in Fig. 1.
MAPKs were identified by virtue of their activa-
tion in response to growth factor stimulation of cells
in culture, hence the name MAPKs (Fig. 2) [19–25].
MAPKs have similar biochemical properties,
immuno-cross-reactivities, amino acid sequence, and
ability to in vitro phosphorylate similar substrates.
Maximal MAPK activity requires that both tyrosine
and threonine residues are phosphorylated. This indi-
cates that MAPKs act as switch kinases that transmit
information of increased intracellular tyrosine phos-
phorylation to that of serine/threonine phosphoryla-
tion [26–28].
Although MAPK activation was first observed in
response to the activation of epidermal growth factor
(EGF), platelet-derived growth factor (PDGF), nerve
growth factor (NGF), and insulin and insulin-like
receptors, other cellular stimuli such as T-cell activa-
tion (which signals through the Lck TyrK); phorbol
esters (which function through activation of protein
kinase C, or PKC); thrombin, bombesin, and brady-
ies of phosphorylation/dephosphorylation steps regulated by kinases
urs in response to a stimulus over a prespecified period of time.
Fig. 2. The modules and various components of the MAPK signaling pathways. The cellular response to growth factors, inflammatory
cytokines, and other mitogens is often mediated by receptors that either are G-protein-linked or are intrinsic protein tyrosine kinases. The
binding of the ligand to receptor tyrosine kinases induces dimerization and autophosphorylation (activation) of the kinase. The activated tyrosine
kinase binds to, and phosphorylates, an adaptor protein, such as Grb2, which, in turn, activates a guanine nucleotide exchange factor, such as
mSOS, which, in turn, activates a small GTP-binding protein, such as Ras or Rac. The GTP-binding proteins then transmit the signal to one of
several cascades of protein Ser/Thr kinases that utilize the sequential phosphorylation of kinases to transmit and amplify the signal. These kinase
cascades are collectively known as mitogen-activated protein kinase (MAPK) signaling cascades. The best studied of these kinase cascades is
the MAPKERK (MAPKp44/p42) signaling cascade. Downstream targets of MAPKERK include p90rsk (p90 risobomal S6 protein kinase) and the
Elk-1 and Stat3 transcription factors. The Jun kinase (MAPKJNK/SAPK) and MAPKp38 kinase pathways are stress-activated MAPK cascades. The
MAPKJNK cascade is activated by inflammatory cytokines as well as by heatshock and UV irradiation. Downstream targets of MAPKJNK
include the transcription factors c-Jun and ATF-2. The MAPKp38 pathway is activated by bacterial endotoxins, inflammatory cytokines, and
osmotic stress. Downstream targets of MAPKp38 include the transcription factors ATF-2, Max, and CREB. MAPKp38 is also involved in the
phosphorylation and activation of heatshock proteins.
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–12851252
kinin (which function through G-proteins); as well as
N-methyl-D-aspartate (NMDA) receptor activation
and electrical stimulation rapidly induce tyrosine
phosphorylation of MAPKs [20,22–24,26–28].
MAPKs are, however, not the direct substrates for
receptor tyrosine kinases (RTKs) or receptor-associ-
ated TyrKs, but are in fact activated by an additional
class of kinases termed MAPK kinases (MKKs) and
MAPK kinase kinases (MAPKK kinases). One of the
MAPKs has been identified as the proto-oncogenic
serine/threonine kinase, Raf [29–42]. Ultimate targets
of the MAPKs are several transcriptional regulators
such as serum response factor (SRF) and the proto-
oncogenes Fos, Myc, and Jun, as well as members of
Table 1
An overview of MAPKs and downstream transcription factors and
substrates
Kinase Substrates
MAPKERK Elk-1, SAP-1, Mnk-1/2,
MAPKAP-K1/p90Rsk, MSK-1
MAPKJNK/SAPK c-Jun, ATF-2, Elk-1
MAPKp38 ATF-2, Elk-1, SAP-1, CHOP,
MEF2C, MAPKAP-K2/K3,
Mnk-1/2, MSK-1, PRAK
MAPKAP-K1/p90Rsk c-Fos, SRF
MSK-1 CREB, histone H3 and HMG-14
For a detailed description, refer to the text.
ATF, activating transcription factor; CHOP, C/EBP homologous
protein; CREB, cAMP response element-binding protein; HMG-14,
high-mobility group-14; MAPKERK, mitogen-activated protein
kinase/extracellular signal-regulated kinase; MAPKAP-K, MAPK-
activated protein kinase; MEF2C, myocyte enhancer factor 2C;
Mnk, MAPK-interacting protein kinase; MSK, mitogen- and stress-
activated protein kinase; PRAK, p38-related/activated protein
kinase; Rsk, ribosomal S6 kinase; SAP-1, SRE accessory protein-
1; SRF, serum response factor.
For a more comprehensive and consistent nomenclature of the
MAPK extended family, refer to Widmann et al. [273].
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–1285 1253
the steroid/thyroid hormone receptor superfamily of
proteins [19,22,23,25–28,31–33,35–37,39–41,43].
The simplified core of a MAPK cascade consists of
three protein kinases: a MAP kinase kinase kinase
(MAPKKK), a MAP kinase kinase (MAPKK), and
MAPK; these kinases phosphorylate each other in
sequence. When activated, MAPKKK phosphorylates
the MAPKK at one or two phosphorylation sites,
bringing activation of the MAPKK module. MAPKKs
are dual-specificity protein kinases that phosphorylate
the MAPK at two phosphorylation sites (almost
always a threonine and a tyrosine residue), bringing
about activation of the MAPK. The active MAPK can
then phosphorylate a variety of target proteins
throughout the cytoplasm and nucleus. On the internal
regulatory mechanisms, each of the kinases in the
MAPK cascade is opposed by one or more phospho-
protein phosphatases. Thus, for an active MAPKK to
activate a MAPK, for example, the rate of MAPK
phosphorylation must exceed the rate of MAPK
dephosphorylation. The activities of these phospha-
tases are high enough to make the output of an MAPK
cascade depend upon the continual presence of a
stimulus feeding into the cascade; if this particular
stimulus is withdrawn, for instance, the downstream
kinases become inactivated within a very short lapse
of time [25,27,36,37,39,40].
The best-characterized vertebrate MAPKs fall into
three subgroups (see Fig. 2). The first subgroup
includes the founding members of the MAPK family,
extracellular signal-regulated kinase-1 (ERK1 or
MAPKERK1/p44), and ERK2 (or MAPKERK2/p42), and
their closest relatives [44–48]. This subgroup is often
referred to as ERKs, although some ERK proteins are
not in fact members of this subgroup family. The
second subgroup is the Jun N-terminal kinases
(JNKs), so called because they can activate the Jun
transcription factor by phosphorylating two residues
near its N-terminus [49–60]. The third subgroup is
the p38 MAPKs, so named because of the molecular
weight (38 kDa) of the first representative of the
subgroup to be discovered [61–68].
Members of both the MAPKJNK and MAPKp38
pathways are also classified as stress-activated protein
kinases (SAPKs) because they are activated in re-
sponse to osmotic shock, UV irradiation, inflammatory
cytokines, and other stressful conditions. In all three
subgroups, a large number of MAKKKs feed into the
activation of a smaller number of MAPKKs and
MAPKs. The diversity of the MAPKKKs thus allows
a wide variety of upstream receptors to couple to
MAPK cascades (see Fig. 2) [35,69–72]. MAPKs
and their corresponding substrates are given in Table 1.
2.2. MAPK signaling as viewed through receptor and
nonreceptor coupled cofactors
The MAPKs are a group of closely related families
of Ser/Thr kinases involved in regulating growth,
differentiation, and cellular responses to stress or
inflammatory cytokines. Whereas the MAPKERK
pathway essentially regulates growth, proliferation,
and differentiation signals, the MAPKJNK/SAPK and
MAPKp38 pathways regulate cell responses to envi-
ronmental stress. The MAPKs link signals generated
at the cell surface to transcription factors via a cascade
of phosphorylation events basically initiated at the
level of the membrane by protein tyrosine kinases
(PTKs), of which two classes exist: the receptor PTKs
and the nonreceptor PTKs. Binding of a ligand to
receptor PTK activates the kinase by dimerization and
autophosphorylation of specific tyrosine sites on the
receptor’s intracellular domain. Among the PTKs is
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–12851254
the Src family of nonreceptor PTKs composed of at
least nine members. Most are restricted to defined
cellular lines, but three (Src, Fyn, and Yes) are
ubiquitously expressed [73]. Like their receptor PTK
counterparts, the Srcs are at the upper end of the
MAPK cascade and serve as initiators for the trans-
duction of signals generated by receptors at the cell
surface. PTK targets contain a 100-amino-acid mod-
ule SH-2 (Src homology 2) and a 50-amino-acid SH-3
module, which recognizes proline-rich domains [74].
The SH-2 site recognizes tyrosine phosphorylation
sites on receptors as well as on nonreceptor proteins
[75].
The next step in the activation of the cascade is the
binding to, and phosphorylation of, an adaptor protein.
Here, two modes have been identified. In one system,
phosphotyrosine binds directly to the SH-2 site on
Grb2. In the other system, the receptor phosphotyr-
osine binds to an intermediary adaptor protein Shc,
which is subsequently bound to Grb2. Shc, a protein
that is widely expressed, is believed to be regulated by
translocation from a cytosolic to a plasma membrane
location where it recruits Grb2 [76]. Integrins, for
example, activate the MAPKERK pathway by means of
an Shc signal. The G-protein-linked thrombin receptor
also uses the Shc adapter to transduce mitogenic
signaling [77]. The Shc protein also mediates the
MAPK response to heat shock response in liver
[78]. Shc signaling is utilized by gastrin (in conjunc-
tion with Ca2 + and PKC) to activate the MAPKERK1
[79]. Endothelin induces Shc association with Grb2 in
glia [80]. Shc binding occurs in response to NGF and
EGF in dorsal root ganglion cells [81]. Shc levels are
low in the brain; however, other predominantly neural
isoforms have been identified: Shc-B, Shc-C, Sck, and
n-Shc [82,83]. The latter two are localized exclusively
in the brain, with considerable overlap in their expres-
sion profiles. The n-Shc variant contains only one
high-affinity Grb2-binding site, while Shc has two
[84,85]. Grb2 is subsequently complexed with a
guanine nuclear exchange factor (GEF).
Two such GEF families have been identified: Sos
and Ras-GRF [86,87]. Proteins of the Sos family are
ubiquitously expressed, while the Ras-GRF class are
expressed primarily in neuronal tissues and are acti-
vated by calcium/calmodulin binding as well as by
phosphorylation. Ras-GRF-2 is more widely ex-
pressed, but is also Ca2 +-regulated. It appears to be a
bifunctional GEF in that it activates the Rho family
proteins in addition to Ras. Sos and Ras-GRF activate
several of the Ras family proteins, but Ras-GRF
uniquely activates a functionally distinct class, R-Ras
GTPase. Sos usually complexes with Grb2 to activate
Ras. The Ras superfamily is comprised of GTPases
that function as molecular switches and exist in the
inactive GDP-bound state or the activated GTP-bound
form. The GEF proteins activate Ras, while another
class of proteins, called GTPase-activating proteins
(GAPs), deactivates Ras by reverting it to the GDP-
bound state [84,85,87].
The Ras G-proteins mediate the mitogen-induced
activation of MAPKs by activating Raf, a family of
serine/threonine kinases. Three kinases comprise the
Raf family: A-Raf, B-Raf, and Raf-1 [88]. In
addition to being activated by the Grb2/Sos/Ras
system, Raf also acts in PKC activation of the
MAPKERK cascade. It has been reported that PKC
activates C-Raf by phosphorylating serine and that
phosphatidate directly activates c-Raf through a
diacylglycerol! PKC! phosphatidic acid! c-Raf
pathway. Protein kinase A (PKA) has recently been
shown to negatively regulate the stimulation of
growth factor responses by directly phosphorylating
Raf-1 at an undetermined site [89]. MEK1 and
MEK2 are the dominant Raf effector proteins, al-
though other Raf substrates have been identified
[90]. Activation of MEK1/2 occurs by phosphoryla-
tion of serine residues at positions 217 and 221,
located in the loop of subdomain VIII. Activation of
MAPKERK1/2 proceeds by phosphorylation of threo-
nine 202 and tyrosine 204.
Deactivation of the MAPKs is an important regula-
tory feature of the cascade. This is accomplished by
phosphatases such as MKP-1/2 PAC-1, which dephos-
phorylates MAPKERK in the nucleus, or MPK-3, which
dephosphorylates cytosolic MAPKERK [91]. The
MAPKs therefore are a convergence point for a wide
variety of extracellular signals. MAPK circuits are a
three-tiered module consisting of Raf!MEK!MAPKERK conveying signals from receptor TKs and
G-protein-coupled receptors to transcription factor in
the cell nucleus, such as p90-Rsk, c-Myc, Elk-1, and
SAP-1. Other targets of MAPKERK1/2 include myelin
basic protein [92] and microtubule-associated protein
[93]. Two other signaling cascades are part of the
MAPK system. The stress-induced MAPKp38 (also
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–1285 1255
known as HOG in the yeast) mediates inflammatory or
stress responses to cytokines, such as TNF, genotoxic
agents, osmotic shock, bacterial lipopolysaccharide,
and photodamage from ultraviolet light as well as from
growth factor withdrawal. The other pathway,
MAPKJNK/SAPK-1, transduces several stress signals
including oxidation/DNA damage along with growth
and differentiation signals. Both of these pathways are
activated by Rac-1 and Cdc-42 signals, which can also
activate the MAPKERK1/2 system. The three MAPK
pathways do not act in isolation but in parallel with
limited cross-talk possible (Fig. 3). MKK kinase
(MEKK)-3 directly activates both MKK-6 (activator
of MAPKp38) and MKK-7 (activator of MAPKJNK),
while MKK-4 (activator of MAPKJNK and MAPKp38)
is activated byMEKK-2 andMEKK-3. TheMAPKJNK
Fig. 3. An overview of the complex network of MAPK signaling pathway
inhibition as ( ); the solid arrows indicate activation (stimulation); P, p
intermediate relaying signals from PKC to C-Raf in the pathway. Abbrev
pathway activates the tumor-suppressor gene, p53,
which has recently been demonstrated to counteract
Cdc-42 [94].
Multiple isoforms of MAPKp38 have been identi-
fied: p38 (p38-a; SAPK-2a), p38-h (SAPK-2b), p38-
h2 (in brain), p38-g (MAPKERK6; SAPK-3), and
p38-y (SAPK-4). The p38-a and p38-h isoforms
are ubiquitously expressed. The p38-g predominates
in muscles, while p38-y is prominent in the lung,
kidney, testis, pancreas, and small intestine [95]. Of
the various isoforms of MAPKp38, only p38-a and
p38-h are sensitive to inhibition by SB-203580.
Several activation routes converge on p38. The
Rac-1 and Cdc-42 signals activate a family of mixed
lineage kinases (MLKs), which further activate MAP
kinase kinases (MKKs). Rac-1 activates MEKK-1 and
s and their interactions and bifurcations. The sign ( ) denotes
hosphorylation. NB: Phosphatide indicates a lipid or phospholipid
iations are listed and defined in the text.
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–12851256
MEKK-2, which subsequently activate MKK-4 that
phosphorylates MAPKp38. MKK-4 mRNA is widely
expressed in murine tissues, but is most abundant in
the muscles and brain [96]. Another route for
MAPKp38 activation is through MKK-6 (MAPKK-6;
MKK-6). MKK-6 (and its variant MKK-6b) activates
p38-a, p38-h2, and p38-g, but p38-y is not activated
by MKK-6. This pathway is activated by Rac-1 and
Cdc-42 signals via MLKs and also transduces TGFhsignals from the TGFhR1 receptor complex via Tak-1,
as well as by Ask-1, a transducer of signals from
FasL, inflammatory cytokines, or stressful insults
such as UV irradiation or apoptotic signals. MKK-3
activates p38-a, p38-h, p38-g, and p38-y [95,96].
MKK-6 activates only the p38-h2 isoform. Addition-
ally, MKK-7, like MKK-6 and MKK-4, phosphory-
lates p38-a at the tyrosine of a Thr–Gly–Tyr motif,
but does not phosphorylate the other MAPKp38 iso-
forms (see Fig. 3).
The individual MAPKp38 isoforms are activated
by specific stimuli and in turn act on a wide range of
substrates. The p38-a isoform is activated by cellular
stress, TNF-a, IL-1h, fMLP, PAF lipopolysacchar-
ides, anisomycin, IL-3, and CD-40l [96,97]. Its sub-
strates are: ATF-2; MAPKAPs 2, 3, and 5; Sap1-a;
CHOP-1 Elk-1 MEF-2C; MSK-1; MBP; PRAK;
P47phox; and MNK-1 [95–98]. The p38-h2 isoform
is activated by cellular stress, TNF-a, IL-1h, UV,
NaCl, and anisomycin, and its substrates are ATF-2,
MAPKAPs 2 and 3, PRAK, and Sap-1a. The p38-g
and p38-y isoforms share similarities in activation
responses and substrates. Both are activated by os-
motic changes PMA IL-1h and TNF-a, but p38-yalso responds to cellular stress. Both kinases act upon
the same set of substrates: ATF-2, stathmin, Elk-1,
Sap-1a, and MBP. Other substrates for MAPKp38 are
MSK-1 and the cytoplasmic Tau proteins [98–100].
The transcription factor c-Jun mediates cell stress
responses [100,101]. The activity of c-Jun is regulated
by phosphorylation of its N-terminal region. The
MAPKJNK is encoded by three genes jnk1 and jnk2,
which are ubiquitously expressed, and jnk3, which is
restricted to the brain, heart, and testis. MAPKJNK
exists in a series of alternatively 3V-spliced variants.
Alternative splicing of MAPKJNK1 and MAPKJNK2
results in two classes by size: 46 kDa (JNK-1b, JNK-
2b) and 55 kDa (JNK-1a, JNK-2a). A second splicing
alternative involving exons in the kinase subdomains
IX and X yields additional variants JNK1-a1, JNK1-
a2, JNK1-b1, JNK1-b2, JNK2-a1, JNK2-a2, JNK2-b1,
and JNK2-b2. The region producing these different
variants is a 23-amino-acid segment between positions
208 and 230 in the protein’s C-terminal region abutting
the catalytic domain of the enzyme. Alternative
MAPKJNK3 splicing yields only a pair of variants 49
kDa (JNK-3b) and 57 kDa (JNK-3a) [101].
Activation of MAPKJNK1 and MAPKJNK2 requires
phosphorylation of threonine at position 183 and
tyrosine at position 185, while for MAPKJNK3 activa-
tion, threonine at position 221 and tyrosine at position
223 are phosphorylated. MKK-4 and MKK-7 syner-
gistically activate MAPKJNK by phosphorylating at
Thr-183, Tyr-185, Thr-404, and Ser-407. MKK-4
preferably phosphorylates Tyr-185 on the Thr–Pro–
Tyr motif on subdomain VII [101]. MKK-7 encodes a
reading frame of 347 amino acids with 11 kinase
subdomains [102]. MKK-7 exists in two variants:
MKK-7a, which preferentially phosphorylates Thr-
183, and MKK-7h, which is equally specific for Thr-
183 and Tyr-185. MKK-7h is hundredfold more
efficient at phosphorylating all three MAPKJNK iso-
forms than MKK-7a [103]. Thr-404 and Ser-407 are
additional phosphorylation sites for MKK-7 action.
MEKK-2 and -3 activate MKK-7 and MKK-4, two
alternative routes leading to MAPKJNK activation.
Recently, a new ubiquitously expressed 898-resi-
due kinase, DPK, has been described, which shows
homology with MEKKs 1–5 and p21-activated ki-
nase (PAK), but no GTPase binding. DPK activates
MAPKERK1/2 and MAPKJNK, but not MAPKp38
[104]. Like in the MAPKp38 pathway, Rac and Cdc-
42 initiate the cascade leading to MAPKJNK activa-
tion, in response to cytokines or stressful stimuli. The
MAPKJNK system is also activated by ceramide via
Tak-1 and by TNF-a via Ask-1 signals, both activat-
ing MKK-4 and MKK-7 [100]. Cadmium activation
of MAPKJNK is mediated by MKK-7 [100,101].
Negative regulators of MAPKJNK have also been
described. In the central nervous system, the excito-
toxin kainic acid downregulates MAPKJNK1. Glutathi-
one S-transferase Pi (GSTp) is an endogenous inhibitor
of MAPKJNK. GSTp is associated with MAPKJNK
under baseline conditions and its dissociation from
MAPKJNK leads to disinhibition. Another class of
negative regulator is the MAPK phosphatases
(MKP), which antagonize MAPKJNK by dephosphor-
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–1285 1257
ylating MAPKJNK as well as its substrate. There are
three isoforms of MKP: MKP-1, which recognizes
MAPKERK, MAPKp38, and MAPKJNK; MKP-2, which
recognizes MAPKERK and MAPKJNK; and MKP-3,
which is specific for MAPKERK [100]. The heat shock
protein, Hsp-72, is also an inhibitor of MAPKJNK
[105].
The translocation of activated MAPKJNK is regu-
lated by a class of proteins called JNK-interacting
proteins (JIPs). Several JIP isoforms have been iden-
tified: JIP-1, expressed in the brain, kidney, and
elsewhere; JIP-2 and JIP-2a; and JIP-3, expressed
exclusively in the brain. JIP-1 contains a phosphotyr-
osine-interacting domain as well as an SH-3 domain.
JIP inhibits the action of MAPKJNK by binding and
retaining MAPKJNK in the cytoplasm [101,106].
MAPKJNK acts upon both nuclear and cytoplasmic
substrates, but does not have as wide a repertoire as
MAPKp38. MAPKJNK activates the transcription fac-
tors c-Jun, ATF-2, and Elk-1, while NFAT-4 is
inactivated. MAPKJNK also inhibits the transcription-
al effect of glucocorticoid receptor; the antiapoptosis
cofactor, Bcl-2, is also deactivated. MAPKJNK phos-
phorylates Tau protein, neurofilaments, and MADD
[100,107]. Additionally, MAPKJNK regulates p53,
which in turn regulates Cdc-42 activity as well as
operates in apoptosis. These complex MAPK cas-
cades and networks are schematically summarized in
Fig. 3.
2.3. MAPK signaling and redox-mediated regulation
of kinases/phosphatases
As discussed above, MAPKs are key signal-trans-
ducing enzymes that are activated by a wide range of
extracellular stimuli. Although regulation of MAPKs
by a phosphorylation cascade has long been recog-
nized as significant, their inactivation through the
action of specific phosphatases has been less studied.
An emerging family of structurally distinct dual-
specificity serine, threonine, and tyrosine phospha-
tases that act on MAPKs consists of 10 members in
mammals, and members have been found in animals,
plants, and yeast [108].
An early role of oxidants in regulating kinases/
phosphatases involved with MAPK induction/inhibi-
tion was reported in neutrophils [109]. It has been
demonstrated that reactive oxygen species (ROS)
were produced by the oxidase regulate tyrosine
phosphorylation, possibly by alterations in the cellu-
lar redox state. For example, immunoprecipitation of
MAPK indicated that a 42- to 44-kDa polypeptide
was tyrosine-phosphorylated in response to treatment
of cells, either with the oxidizing agent diamide or
with H2O2 in cells where catalase was inhibited.
Furthermore, exposure of cells to oxidants caused a
significant increase in the activity of MEK, as
determined by an in vitro kinase assay using recom-
binant catalytically inactive glutathione S-transferase
MAPK as the substrate. Additionally, oxidant treat-
ment of cells resulted in inhibition of the activity of
CD45, a protein tyrosine phosphatase known to
dephosphorylate and inactivate MAPK, thereby con-
cluding that oxidant treatment of neutrophils can
activate MAPK by stimulating its tyrosine and,
presumably, threonine phosphorylation via MEK
activation, a response that may be potentiated by
inhibition of MAPK dephosphorylation by phospha-
tases such as CD45 [109].
The aforementioned observations were also reaf-
firmed in muscle cells [110]. Vascular smooth muscle
cell growth, as measured by [3H]thymidine incorpo-
ration, was stimulated by H2O2 and the naphtho-
quinolinedione LY83583. There was an increase in
MAPK activity by LY83583 but not by H2O2. In
addition, activation of MAPK by LY83583 was
PKC-dependent. Of note, expression of MAP kinase
phosphatase-1 (MKP-1), a transcriptionally regulated
redox-sensitive protein tyrosine/threonine phospha-
tase, showed that although H2O2 induced MKP-1
mRNA to a greater extent than did LY83583, the
increased MKP-1 expression could not explain the
inability of H2O2 to stimulate MAPK because
mRNA levels were not detected until an hour later,
thereby indicating that additional signal events are
required for the mitogenic effects of H2O2 [110].
Moreover, the thiol-depleting agents, phenylarsine
oxide (a tyrosine phosphatase inhibitor) and N-ethyl-
maleimide, were reported to inhibit the phorbol ester-
induced PKC activation in vascular smooth muscle
cells [111]. In addition, sodium orthovanadate, also a
protein tyrosine phosphatase inhibitor, could neither
activate nor inhibit PKC, suggesting that oxidation of
the cellular thiols inhibits PKC and activates MAPK,
indicating that the activation of MAPK is indepen-
dent of PKC.
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–12851258
In mesangial cells, redox- and oxidant-dependent
assessment of MAPK identified a rapid and delayed
phase of activation. For instance, rapid and late
MAPK activations were attenuated by the redox-
modulating agent, N-acetylcysteine [112]. Specifical-
ly, late-phase activation coincided with endogenous
nitric oxide (NO) generation and in turn was sup-
pressed by the NO synthase-blocking compound
diphenyleniodonium or nitroarginine methyl ester.
Of particular interest, late and persistent MAPK
activation, induced by NO donors or endogenously
generated NO, was found in association with inhi-
bition of phosphatase activity. Moreover, in vitro
dephosphorylation of activated and immunoprecipi-
tated p42/p44 by cytosolic phosphatases was sensi-
tive to the readdition of NO and was found to be
inhibited in the cytosol of S-nitrosoglutathione-stim-
ulated cells. Conclusively, NO affects MAPKERK
twofold: rapid activation is cGMP-mediated, where-
as late activation is transmitted via inhibition of
tyrosine dephosphorylation [112]. In concert, it has
been demonstrated that a redox-sensitive protein
phosphatase activity regulates the phosphorylation
state of MAPKp38 in primary astrocyte culture,
suggesting that ROS are used as second-messenger
substances that activate MAPKp38 in part via the
transient inactivation of regulatory protein phospha-
tases [113]. On the particular mechanism involved
in redox-mediated regulation of MAPKs, it has been
suggested that the differential interaction of the
tyrosine phosphatases PTP-SL, STEP, and HePTP
with MAPKERK and MAPKp38a could be deter-
mined by a kinase specificity sequence and influ-
enced by reducing agents [114], indicating that
intracellular redox conditions could modulate the
activity and subcellular location of MAPKs by
controlling their association with their regulatory
PTPs. A closely related mechanism was reported
for cell adhesion. It was indicated that ROS are
essential mediators of cell adhesion; specifically, the
oxidative inhibition of a tyrosine phosphatase is
required for cell adhesion. This signaling network
unraveled a redox circuitry whereby, upon cell ad-
hesion, oxidative inhibition of a protein tyrosine
phosphatase promotes the phosphorylation/activation
and the downstream signaling MAPKs and, as a
final event, cell adhesion and spreading onto fibro-
nectin [115].
3. Hypoxia-mediated regulation of MAPK
signaling pathways
3.1. Hypoxia and oxygen-sensing mechanisms
How do organisms sense the level of oxygen in the
environment/microenvironment and respond appropri-
ately when oxygen level decreases (a condition
termed hypoxia) [9,10,19,112,116,117]? The expres-
sion of genes is predominantly determined by con-
ditions of the cell microenvironment. Prime examples
of such regulation are found in embryonic develop-
ment of all multicellular organisms. 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 such as those
occurring during the birth transition period. Upon
ligand binding, this presumably membrane-bound
receptor transduces intracellular chemical/redox sig-
nals that relay messages for the regulation of gene
expression, a phenomenon mainly involving the acti-
vation of transcription factors, ion channels, and
chemoreceptors (Fig. 4).
Oxygen sensing and the underlying molecular
stratagems have been the focus of experimental inves-
tigations trying to find an answer to the question:
‘‘What is the identity of the oxygen sensor?’’ [5–8].
The original proposed molecular mechanism underly-
ing oxygen sensing in mammalian cells involves an
oxygen sensor that is a heme protein. Studies on
erythropoietin (EPO), a glycoprotein hormone re-
quired for the proliferation and differentiation of
erythroid cells, demonstrate that EPO production is
enhanced under hypoxic conditions. Furthermore, the
induction of EPO expression by transition metals such
as cobalt (Co2 +) and nickel (Ni2 +) supports the
hypothesis that the oxygen sensor for the induction
of this glycoprotein is a heme protein and that these
metal atoms can substitute for the iron atom within the
heme moiety [5,6,118,119].
Further evidence supporting the notion that the
oxygen sensor is a heme protein came with additional
studies that utilized carbon monoxide (CO); CO can
noncovalently bind to ferrous (Fe2 +) heme groups in
hemoglobin, myoglobin, cytochromes, and other
heme proteins [5,6], where its ligation state is struc-
Fig. 4. Oxygen-sensing mechanisms during hypoxia involve the membrane-bound NADPH oxidase, the mitochondria–cytochrome complex
system (A), and chemoreceptors (B), such as K+ channels. This ultimately leads to sensory recognition and activity upregulation.
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–1285 1259
turally identical to that of oxygen. It was subsequently
proposed that the effect of CO on oxygen sensing
might occur via locking of the sensor in an oxy-
conformation, which could involve a multisubunit
mechanism [118–120].
In addition to the aforementioned models for
oxygen sensing, certain pharmacological studies, led
by Fandrey et al. [121], suggest that the oxygen sensor
might involve a microsomal mixed function oxidase.
Based on these studies, it was proposed that oxygen
sensing for EPO involves an interaction between
cytochrome P450 and cytochrome P450 reductase,
thereby allowing the conversion of molecular oxygen
to superoxide anion (O2�) and H2O2 radicals [5–
8,119–122]. Acker [123] has provided support for
the central role of an oxidase in oxygen sensing based
on spectroscopic evidence. It was reported that b-
cytochrome functions as a NAD(P)H oxidase, con-
verting oxygen to O2�. The enzymatic complex in
mammalian cells is membrane-bound and transduces
the conversion of molecular oxygen to ROS, accord-
ing to the following equations:
CytFe2þ þ O2 ! CytFe2þO2
CytFe2þO2 ! CytFe3þ þ O�:2
CytFe3þ þ NADðPÞH ! CytFe2þ þ NADðPÞþ
A resurgence of interest in mitochondrial physiol-
ogy has recently developed as a result of new exper-
imental data demonstrating that mitochondria function
as important participants in a diverse collection of
novel intracellular signaling pathways. Further experi-
ments showed a potential involvement of the mito-
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–12851260
chondria in oxygen sensing [124]. For instance, a
spectroscopic photolysis with monochromatic light
has identified a CO-binding heme protein falling
within the spectrum of the mitochondrial cytochrome
a3 [125]. It was consequently proposed that this heme
protein, presumably located on the plasma membrane,
has a low affinity for oxygen and a relatively high
affinity for CO. The same model predicted that
another heme protein in the mitochondria has a
relatively higher affinity for oxygen and a lower
affinity for CO. The biochemical reaction, which
was proposed as an alternative way of regenerating
ferroheme in the oxygen sensor, is given below:
COþ 2Fe3þ þ H2O ! CO2 þ 2Fe2þ þ 2Hþ
These aforementioned observations pertaining to
the mitochondrion as a possible oxygen sensor were
unequivocally supported by novel studies recently
reported. Cardiomyocytes are known to suppress con-
traction and oxygen consumption during hypoxia.
Cytochrome oxidase undergoes a decrease in Vmax
during hypoxia, which could alter mitochondrial redox
status and increase the generation of ROS. Duranteau
et al. [126] tested whether ROS generated by mito-
chondria act as second messengers in the signaling
pathway linking the detection of oxygen with the
functional response. In this respect, contracting cardi-
omyocytes were superfused under controlled oxygen
conditions, while fluorescence imaging of 2,7-dichlor-
ofluorescein (DCF) was used to assess ROS genera-
tion. Compared with normoxia, graded increases in
DCF fluorescence were seen during hypoxia. In addi-
tion, the antioxidants 2-mercaptopropionyl glycine
and 1,10-phenanthroline attenuated these increases
and abolished the inhibition of contraction. Super-
fusion of normoxic cells with H2O2 mimicked the
effects of hypoxia by eliciting decreases in contraction
that were reversible. To test the role of cytochrome
oxidase, sodium azide was added during normoxia to
reduce the Vmax of the enzyme. It was observed that
azide produced graded increases in ROS signaling,
accompanied by graded decreases in contraction that
were reversible, demonstrating that mitochondria re-
spond to graded hypoxia by increasing the generation
of ROS and suggesting that cytochrome oxidase may
contribute to this oxygen-sensing mechanism [126].
The same group also reported that mitochondrial
ROS trigger hypoxia-induced transcription. Chandel
et al. [127] tested whether mitochondria act as oxy-
gen sensors during hypoxia, and whether hypoxia and
CO activate transcription by increasing the generation
of ROS. Results showed that: (i) wild-type Hep3B
cells increased ROS generation during hypoxia or
CoCl2 incubation; (ii) Hep3B cells depleted of mito-
chondrial DNA (U0 cells) failed to respire; failed to
activate mRNA for EPO, glycolytic enzymes, or
vascular endothelial growth factor (VEGF) during
hypoxia; and failed to increase ROS generation
during hypoxia; (iii) U0 cells increased ROS genera-
tion in response to CoCl2 and retained the ability to
induce expression of these genes; and (iv) the anti-
oxidants pyrrolidine dithiocarbamate (PDTC) and
ebselen, a glutathione (GSH) peroxidase mimetic,
abolished transcriptional activation of these genes
during hypoxia or CoCl2 in wild-type cells and
abolished the response to CoCl2 in U0 cells [127]. It
was proposed that hypoxia activates transcription via
a mitochondria-dependent signaling process involv-
ing increased ROS, whereas CoCl2 activates tran-
scription by stimulating ROS generation via a
mitochondria-independent mechanism [126–129].
In another interesting observation, Chandel et al.
reported that mitochondrial ROS play a major role in
HIF-1a regulation. In this respect, it was observed that
hypoxia increased mitochondrial ROS generation at
complex III, which caused the accumulation of HIF-
1a protein responsible for initiating expression of a
luciferase reporter construct under the control of a
hypoxic response element [130]. Of note, this re-
sponse was lost in cells depleted of mitochondrial
DNA. Furthermore, overexpression of catalase abol-
ished hypoxic response element luciferase expression
during hypoxia. In addition, exogenous H2O2 stabi-
lized HIF-1a protein during normoxia and activated
luciferase expression in wild-type and U0 cells. In fact,isolated mitochondria increased ROS generation dur-
ing hypoxia, indicating that mitochondria-derived
ROS are both required and sufficient to initiate HIF-
1a stabilization during hypoxia, thereby implicating
this transcription factor as a possible oxygen sensor
(see below).
A nonmitochondrial oxygen sensor has, however,
been recently proposed. Ehleben et al. applied bio-
physical methods, such as light absorption spectropho-
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–1285 1261
tometry of cytochromes, determination of NAD(P)H-
dependent O2� formation, and localization of �OH by
three-dimensional (3D) confocal laser scanning mi-
croscopy, to reveal putative members of the oxygen-
sensing signal pathway leading to enhanced gene
expression under hypoxia [131,132]. A cell mem-
brane-localized nonmitochondrial cytochrome b558
seemed to be involved as an oxygen sensor in the
hepatoma cell line HepG2 in cooperation with the
mitochondrial cytochrome b563, probably probing
additionally metabolic changes. The hydroxyl radical,
a putative second messenger of the oxygen-sensing
pathway generated by a Fenton reaction, could be
visualized in the perinuclear space of the three human
cell lines used. Substances like cobalt or the iron
chelator desferrioxamine, which have been applied in
HepG2 cells to mimic hypoxia-induced gene expres-
sion, interact on various sides of the oxygen-sensing
pathway, confirming the importance of b-type cyto-
chromes and the Fenton reaction.
NADPH oxidase isoforms, with different gp91phox
subunits as well as an unusual cytochrome aa3 with a
heme a/a3 relationship of 9:91, were discussed as
putative oxygen sensor proteins influencing gene ex-
pression and ion channel conductivity [133]. ROS are
believed to be important second messengers of the
oxygen-sensing signal cascade determining the stabil-
ity of transcription factors or the gating of ion
channels. The formation of ROS by a perinuclear
Fenton reaction was imaged by one- and two-photon
confocal microscopy revealing mitochondrial and
nonmitochondrial generation. In reference to the
aforementioned observation, some recent concepts
on oxygen-sensing mechanisms at the carotid body
chemoreceptors were highlighted [134]. Most avail-
able evidence suggested that glomus (type I) cells are
the initial sites of transduction, and they release
transmitters in response to hypoxia, which in turn
depolarize the nearby afferent nerve ending, leading
to an increase in sensory discharge.
Two main hypotheses have been advanced to ex-
plain the initiation of the transduction process that
triggers transmitter release. One hypothesis assumed
that a biochemical event associated with a heme protein
triggers the transduction cascade. Supporting this idea,
it has been shown that hypoxia might affect mitochon-
drial cytochromes. In addition, there was a body of
evidence implicating nonmitochondrial enzymes such
as NADPH oxidases, NO synthases, and heme oxy-
genases located in glomus cells [134]. These proteins
could contribute to transduction via generation of ROS,
NO, and/or CO.
The other hypothesis suggested that a K+ channel
protein is the oxygen sensor, and that inhibition of this
channel and the ensuing depolarization is the initial
event in transduction, as indicated by Peers and Kemp
[135]. Several oxygen-sensitive K+ channels have
been identified. However, their roles in the initiation
of the transduction cascade and/or cell excitability
remain unclear. In addition, recent studies indicated
that molecular oxygen and a variety of neurotrans-
mitters might also modulate Ca2 + channels [134].
Most importantly, it is possible that the carotid body
response to oxygen requires multiple sensors, and
they work together to shape the overall sensory
response of the carotid body over a wide range of
arterial oxygen tensions.
The hypothesis that there exists a specific oxygen
sensor(s), which relay(s) chemical signals intracellu-
larly, is therefore consistent with the notion that there
is a unifying mechanism involved in transducing
dynamic changes in pO2 to the nucleus [5–8]. In
response to DpO2, there is a coordinate expression of
genes needed to confer appropriate responses to
hypoxia or hyperoxia. The regulation of physiologi-
cally important oxygen-responsive and redox-sensi-
tive genes would, therefore, dictate well-controlled
responses of the cell within a challenging environment
and necessarily would determine the specificity of
cellular adaptation.
3.2. Hypoxia and MAPK signaling
In 1997, Seko et al. [136] directly and unequivocally
reported that both hypoxia and hypoxia/reoxygenation
rapidly activate upstream Src family TyrKs and p21ras.
This was followed by the sequential activation of
MAPKKK activity of Raf-1, MAPKK, MAPKs in-
cluding MAPKERK1/2, and S6 kinase (p90-rsk). Fur-
thermore, it was demonstrated that hypoxia and
hypoxia/reoxygenation could cause rapid activation
of stress-activatedMAPK signaling cascades involving
p65-PAK, MAPKp38, and SAPK. These stimuli also
caused the phosphorylation of activating the down-
stream transcription factor (ATF)-2 [137]. Because
p65-PAK is known to be upstream of MAPKp38 and
Fig. 5. Hypoxia-mediated regulation of MAPK signaling pathways.
The role of the different MAPK signaling pathways in regulating
cellular responses, including cell survival and death, in response to
incoming signals such as oxygen deprivation (hypoxia).
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–12851262
also a target of p21rac-1, which belongs to the Rho
subfamily of p21ras-related small GTP-binding pro-
teins, these results suggested that two different stress-
activated MAPK pathways distinct from the classical
MAPK pathway were activated in response to hypoxia
and hypoxia/reoxygenation.
In concert, Yu et al. [138] reported that cardiomyo-
cytes subjected to brief episodes of hypoxia possess a
resistance to serious damaging effects exerted by a
subsequent long-time hypoxia on these cells, a condi-
tion that was termed hypoxic preconditioning (PC).
On a model of hypoxia/reoxygenation of cultured
neonatal rabbit cardiomyocytes, the changes of
MAPK and RSK activity were recorded. It was found
that intracellular total MAPK and nuclear MAPK,
after a period of reoxygenation-preceded hypoxia,
were increased [138]. Moreover, intracellular RSK
activity increased by hypoxia/reoxygenation. Phos-
phatase-1 (Ppase-1) inhibitor (okadaic acid, or OA)
augmented the increase of MAPK and RSK activity
induced by hypoxia/reoxygenation. However, TyrK
inhibitor (genistein), PKC inhibitor (H7), and prein-
cubation of cardiomyocytes with PKC activator PMA
all reduced MAPK activation by hypoxia/reoxygena-
tion. In addition, PKA inhibitor (H89), Ca2 +/calmod-
ulin (CaM)-dependent protein kinase (PKM) inhibitor
(W7), or Ppase-2a inhibitor (OA) had no effect on
MAPK and RSK activity, thus indicating that the
activation of MAPK and RSK activity during hypox-
ia/reoxygenation might require the participation of
PKC, TyrK, and Ppase-1, while PKA, PKM, and
Ppase-2a were not involved.
Further elaborating on the mechanisms involved
in hypoxia-mediated regulation of MAPK signaling,
Mizukami et al. [139] recently reported that PKC-a,
an atypical PKC isoform mainly expressed in rat
heart, can act as an upstream kinase of MAPK
during ischemic hypoxia and reoxygenation. Immu-
nocytochemical observations showed PKC-a stain-
ing in the nucleus during ischemic hypoxia and
reoxygenation when phosphorylated MAPK was
also detected in the nucleus. This nuclear localiza-
tion of PKC-a was inhibited by treatment with
Wortmannin. This was supported by the inhibition
of MAPK phosphorylation by another blocker of
phosphoinositide 3-kinase, LY294002. Moreover, an
upstream kinase of MAPK, MEK1/2, was signifi-
cantly phosphorylated after reoxygenation (observed
mainly in the nucleus), whereas it was present in the
cytoplasm in serum stimulation. PKC inhibitors and
phosphoinositide 3-kinase inhibitors, as observed in
the case of MAPK phosphorylation, blocked the
phosphorylation of MEK, indicating that PKC-a,
which is activated by phosphoinositide 3-kinase,
might induce MAPK activation through MEK in
the nucleus during reoxygenation after ischemic
hypoxia.
In parallel, exposure to moderate hypoxia (5% O2)
was found to progressively stimulate the phosphoryla-
tion and activation of different isoforms of MAPKp38,
such asMAPKp38-g, in particular, and alsoMAPKp38-a.
In contrast, hypoxia had no effect on the enzyme
activity of MAPKp38-h, MAPKp38-h2, MAPKp38-y, or
even on MAPKJNK [139]. Prolonged hypoxia also
induced the phosphorylation and activation of MAP-
KERK1/2, although this activation was modest when
compared to NGF and UV-induced activation [140]. It
was also shown that the activation of MAPKp38-g
during hypoxia required calcium (Ca2 +), as treatment
with Ca2 +-free media or the CaM antagonist, W13,
blocked the activation of MAPKp38-g. These studies
demonstrated that an extremely typical physiological
stress could cause selective activation of specific ele-
ments of the SAPKs and MAPKs, and identify Ca2 +/
CaM as a critical upstream activator. The role of
hypoxia in mediating MAPK signaling is schematized
in Fig. 5.
Fig. 6. Hypoxia signal transduction. Reduction of cellular O2
concentration is associated with redox changes that lead to altered
phosphorylation of HIF-1a, which increases its stability and
transcriptional activity, resulting in the induction of downstream
gene expression. Putative inducers (horizontal arrows) and
inhibitors (blocked arrows) of different stages in the proposed
pathway are indicated.
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–1285 1263
4. Hypoxia-mediated regulation of transcription
factors and gene transcription: the role of
MAPK-related signaling pathways
4.1. MAPK-mediated regulation of HIF
4.1.1. HIF and oxygen sensing—an overview
In order to maintain oxygen homeostasis, a process
that is, of course, essential for survival, pO2 delivery
to the mitochondrial electron transport chain must be
tightly maintained within a narrow physiological
range [5–8]. However, this system may fail with
subsequent induction of hypoxia, resulting in either
a failure to generate sufficient ATP to sustain meta-
bolic activities, or a hyperoxic condition that contrib-
utes to the generation of ROS, which, in excess, could
be cytotoxic and often cytocidal. Adaptive responses
to hypoxia involve the regulation of gene expression
by HIF-1a, whose expression, stability, and transcrip-
tional activity increase exponentially on lowering pO2
[141–143].
HIF-1a is a mammalian transcription factor
expressed uniquely in response to physiologically
relevant hypoxic conditions [5–8,141–143]. Studies
of the EPO gene led to the identification of a cis-acting
hypoxia response element (HRE) in the 3V-flankingregion. HIF-1 was identified as a hypoxia-inducible
HRE-binding activity. The HIF-1 binding site was
subsequently used for purification of the HIF-1a and
HIF-1h subunits by DNA affinity chromatography.
Both HIF-1 subunits are basic helix– loop–helix
(bHLH)–PAS proteins: HIF-1a is a novel protein;
HIF-1h is identical to the aryl hydrocarbon receptor
nuclear translocator (ARNT) protein. HIF-1a DNA-
binding activity and HIF-1a protein expression are
rapidly induced by hypoxia and the magnitude of the
response is inversely related to pO2 [142–145].
In hypoxia, multiple systemic responses are in-
duced, including angiogenesis, erythropoiesis, and
glycolysis. HREs containing functionally essential
HIF-1 binding sites are identified in genes encoding
VEGF, glucose transporter-1 (GLUT-1), and the gly-
colytic enzymes aldolase A, enolase-1 (ENO-1), lac-
tate dehydrogenase A (LDH-A), and phosphoglycerate
kinase-1 [5–8]. HIF-1a is an important mediator for
increasing the efficiency of oxygen delivery through
EPO and VEGF. A well-controlled process of adapta-
tion parallels this with decreased oxygen availability
through the expression and activation of glucose trans-
porters and glycolytic enzymes. Of note, EPO is
responsible for increasing blood oxygen-carrying ca-
pacity by stimulating erythropoiesis, VEGF is a tran-
scriptional regulator of vascularization and glycolytic
transporters, and enzymes increase the efficiency of
anaerobic generation of ATP.
HIF-1a has also been shown to activate transcrip-
tion of genes encoding inducible nitric oxide synthase
(iNOS) and heme oxygenase-1 (HO-1)—which are
responsible for the synthesis of the vasoactive mole-
cules NO and CO, respectively—as well as transfer-
rin—which, like EPO, is essential for erythropoiesis.
Each of these genes contains an HRE sequence of
< 100 bp that includes one or more HIF-1 binding
sites containing the core sequence 5V-RCGTG-3V [5–
8]. It is expected that any reduction of tissue oxygen-
ation in vivo and in vitro would therefore provide a
mechanistic stimulus for a graded and adaptive re-
sponse mediated by HIF-1a. Hypoxia signal trans-
duction is schematized in Fig. 6.
Several of the major molecular mechanisms that
regulate HIF-1 have recently emerged to shed a
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–12851264
thorough light on the role of this transcription factor in
oxygen sensing [144,145]. The von Hippel–Lindau
protein (pVHL) has emerged as a key factor in cellular
responses to oxygen availability, being required for the
oxygen-dependent proteolysis of the a-subunits of
HIF (Fig. 7). Mutations in VHL cause a hereditary
cancer syndrome associated with dysregulated angio-
genesis and upregulation of hypoxia-inducible genes
[145]. Recently, Lee et al. [142], Salceda and Caro
[143], Zhu and Bunn [144], and Maxwell and Ratcliffe
[145] unequivocally elaborated on the mechanisms
underlying these processes and showed that extracts
from VHL-deficient renal carcinoma cells have a
defect in HIF-1a ubiquitination activity, which was
complemented by exogenous pVHL. This defect was
specific for HIF-1a among a range of substrates tested.
Furthermore, HIF-1a subunits were the only
pVHL-associated proteasomal substrates identified
by comparison of metabolically labeled anti-pVHL
immunoprecipitates with proteosomally inhibited
cells and normal cells. Analysis of pVHL/HIF-1a
interactions defined short sequences of conserved
residues within the internal transactivation domains
of HIF-1a molecules sufficient for recognition by
pVHL. In contrast, while full-length pVHL and the
p19 variant interacted with HIF-1a, the association
was abrogated by further N-terminal and C-terminal
truncations. The interaction was also disrupted by
Fig. 7. Potential oxygen-sensing mechanisms and the role of the
transcription factor HIF. This schematic shows the role of von
Hippel–Lindau (VHL) tumor-suppressor protein in mediating the
regulation of HIF.
tumor-associated mutations in the h-domain of pVHL
and loss of interaction was associated with defective
HIF-1a ubiquitination and regulation, defining a
mechanism by which these mutations generate a
constitutively hypoxic pattern of gene expression
promoting angiogenesis [145–148]. These findings
clearly indicate that pVHL regulates HIF-1a proteol-
ysis by acting as the recognition component of a
ubiquitin ligase complex and supports a model in
which its h-domain interacts with short recognition
sequences in the a-subunits.
Moreover, in oxygenated and iron-replete cells,
HIF-1a subunits were rapidly destroyed by a mech-
anism that involved ubiquitination by the pVHL E3
ligase complex [149]. This process was suppressed by
hypoxia and iron chelation, allowing transcriptional
activation. Jaakkola et al. [149] recently indicated that
the interaction between human pVHL and a specific
domain of the HIF-1a subunit is regulated through
hydroxylation of a proline residue (HIF-1a P564) by
an enzyme termed by the authors as HIF-a prolyl-
hydroxylase (HIF-PH or HPH). An absolute require-
ment for oxygen as a cosubstrate and iron as a
cofactor suggested that HIF-PH functions directly as
a cellular oxygen sensor. Furthermore, Masson et al.
[150] recently identified two independent regions
within the HIF-a oxygen-dependent degradation do-
main (ODDD), which are targeted for ubiquitination
by VHLE3 in a manner dependent upon prolyl
hydroxylation (Fig. 8). In a series of in vitro and in
vivo assays, Masson et al. demonstrated the indepen-
dent and nonredundant operation of each site in the
regulation of the HIF system. Both sites contain a
common core motif, but differ both in overall se-
quence and conditions under which they bind to the
VHLE3 ligase complex [150]. The definition of two
independent destruction domains implicated a more
complex system of pVHL–HIF–a interactions, but
reinforced the role of prolyl hydroxylation as an
oxygen-dependent destruction signal.
These mechanisms were also reported in lower
invertebrates as potential pathways for HIF oxygen
sensing. For instance, Epstein et al. [151] defined a
conserved HIF–VHL–prolyl hydroxylase pathway in
Caenorhabditis elegans and used a genetic approach
to identify EGL-9 as a dioxygenase that regulates HIF
by prolyl hydroxylation. In mammalian cells, it was
shown that the HIF-prolyl hydroxylases were repre-
Fig. 8. The regulation of HIF by the prolyl hydroxylase enzyme, a
putative oxygen sensor. VHL gene product (pVHL) interacts with
HIF-1a and is required for the destruction of HIF-1a at the ODDD
under normoxic conditions. HIF–pVHL interaction depends on both
oxygen and iron availability. Furthermore, HIF-1a–pVHL interac-
tion requires enzymatic posttranslational hydroxylation of HIF-1a at
a single proline. This prolyl hydroxylation requires, besides oxygen
and iron, also a citric acid cycle intermediate 2-oxoglutarate.
Together with the HIF-induced activation of glucose and iron
metabolism genes, it creates a tight link between oxygen sensing and
cellular control of metabolism. Three novel human prolyl hydrox-
ylases (PHDs) that modify HIF-1a have been characterized. Their
activity is regulated by oxygen, 2-oxoglutarate, and iron availability,
suggesting that PHDs may function as cellular oxygen sensors.
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–1285 1265
sented by a series of isoforms bearing a conserved 2-
histidine-1-carboxylate–iron coordination motif at the
catalytic site. Direct modulation of recombinant en-
zyme activity by graded hypoxia, iron chelation, and
cobaltous ions mirrored the characteristics of HIF
induction in vivo, thereby fulfilling requirements for
these enzymes as oxygen sensors that regulate this
transcription factor [152–155].
Similarly, Bruick and McKnight [156] reported
that the inappropriate accumulation of HIF caused
by forced expression of the HIF-1a subunit under
normoxic conditions was attenuated by coexpression
of HPH. Suppression of HPH in cultured Drosophila
melanogaster cells by RNA interference resulted in
elevated expression of a hypoxia-inducible gene
(LDH; encoding lactate dehydrogenase) under nor-
moxic conditions, indicating that HPH is an essential
component of the pathway through which cells sense
oxygen. In complement with the aforementioned
observations, Lando et al. [157] demonstrated that
the hypoxic induction of the COOH-terminal trans-
activation domain (CAD) of HIF occurs through
abrogation of hydroxylation of a conserved asparagine
(Asn) in the CAD. Inhibitors of Fe2 +- and 2-oxoglu-
tarate-dependent dioxygenases prevented hydroxyl-
ation of the Asn, thus allowing the CAD to interact
with the p300 transcription coactivator. Replacement
of the conserved Asn by alanine (Ala) resulted in
constitutive p300 interaction and strong transcription-
al activity. Moreover, the full induction of HIF,
therefore, possibly relies on the abrogation of both
proline (Pro) and Asn hydroxylation, which, during
normoxia, occurs at the degradation and COOH-
terminal transactivation domains, respectively.
Recently, an oxygen-sensitive cousin of HIF-1 has
been identified, characterized, and cloned. Hypoxia-
inducible factors (HIF-1, HIF-2, and HIF-3) are close-
ly related protein complexes that are oxygen-respon-
sive. The cDNAs of three HIF a-subunits were cloned
from RNA of primary rat hepatocytes by reverse
transcriptase polymerase chain reaction [158]. All
three cDNAs encoded functionally active proteins of
825, 874, and 662 amino acids, respectively. After
transfection, they were able to activate the luciferase
activity of a luciferase gene construct containing three
HIF-responsive elements. The mRNAs of the rat HIF
a-subunits were expressed predominantly in the peri-
venous zone of rat liver tissues; the nuclear HIF-a
proteins, however, did not appear to be zonated [158].
HIFs locate to HIF-binding sites (HBS) within the
HREs of oxygen-regulated genes [159,160]. Whereas
HIF-1a is, generally, expressed ubiquitously, HIF-2a
(EPAS) is found primarily in the endothelium, similar
to endothelin-1 (ET-1) and fms-like tyrosine kinase-1
(Flt-1), the expression of which is controlled by HREs.
Camenisch et al. [161] identified a unique sequence
alteration in both ET-1 and Flt-1 HBS not found in
other HIF-1 target genes, implying that these HBS
might cause binding of HIF-2 rather than HIF-1.
However, electrophoretic mobility shift assays showed
HIF-1 and HIF-2 DNA complex formation with the
unique ET-1 HBS about equal. Both DNA-binding
and hypoxic activation of reporter genes using the ET-
1 HBS were decreased compared with transferrin and
EPO HBS. The Flt-1 HBS, in addition, was nonfunc-
tional when assayed in isolation, suggesting that
additional factors are required for hypoxic upregula-
tion via the reported Flt-1 HRE [161]. Interestingly,
HIF-1 activity could be restored fully by point mutat-
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–12851266
ing the ET-1 (but not the Flt-1) HBS, suggesting that
the wild-type ET-1 HBS attenuate the full hypoxic
response known from other oxygen-regulated genes
[161–163].
4.1.2. HIF and MAPK regulation
Adaptive responses to hypoxia involve the reg-
ulation of gene expression by HIF, whose expres-
sion, stability, and transcriptional activity increase
exponentially on lowering the partial pressure of
oxygen (pO2) [5–8,163]. Tumor angiogenesis, the
development of new blood vessels, is a highly regu-
lated process that is controlled genetically by alter-
ations in oncogene and tumor-suppressor gene
expression and physiologically by the tumor microen-
vironment (hypoxia). Previous studies indicated that
the angiogenic switch in Ras-transformed cells might
be physiologically promoted by the tumor microenvi-
ronment through the induction of the angiogenic
mitogen, VEGF.
HIF-1 controls the expression of a number of genes
such as VEGF and EPO in low-oxygen conditions
[164–167]. However, the molecular mechanisms that
underlie the activation of the limiting subunit, HIF-1a,
are still poorly resolved. In this respect, Mazure et al.
[168] showed that Ras-transformed cells do not use the
downstream effectors c-Raf-1 or MAPK in signaling
VEGF induction by hypoxia, as overexpression of
kinase-defective alleles of these genes did not inhibit
VEGF induction under low-oxygen conditions. In
contrast to the c-Raf-1/MAPK pathway, hypoxia in-
creased phosphatidylinositol 3-kinase activity in a
Ras-dependent manner, and the inhibition of phospha-
tidylinositol 3-kinase activity, genetically and pharma-
cologically, resulted in the inhibition of VEGF
induction [168]. It was proposed that hypoxia modu-
lates VEGF induction in Ras-transformed cells
through the activation of a stress-inducible phosphati-
dylinositol 3-kinase/Akt pathway and the HIF-1 tran-
scriptional response element.
On the mechanism of MAPK-dependent regulation
of VEGF, it was demonstrated that the MAPKERK1/2
signaling cascade controls VEGF expression at two
levels. In normoxic conditions, MAPKs activate the
VEGF promoter at the proximal (� 88/� 66) region
where substance P (Sp)-1/activating protein (AP)-2
factors bind [83]. Activation of MAPKERK1/2 was
sufficient to turn on VEGF mRNA. Furthermore,
MAPKERK1/2 induced the phosphorylation of HIF-1a
in vitro and HIF-1-dependent VEGF gene expression
was strongly enhanced by the exclusive activation of
MAPKERK1/2. Of note, the regulation of MAPKERK1/2
activity was critical for controlling the proliferation
and growth arrest of vascular endothelial cells at
confluency [169]. Taken together, these data pointed
to major targets of angiogenesis where MAPKERK1/2
might exert a determinant action [170,171].
Furthermore, preliminary results showing that en-
dogenous HIF-1a migrated 12 kDa higher than in
vitro-translated protein led Richard et al. [172] to
evaluate the possible role of phosphorylation on this
phenomenon. In this respect, it was reported that HIF-
1a was strongly phosphorylated in vivo and that its
phosphorylation was responsible for the marked differ-
ences in the migration pattern of HIF-1a. In vitro, HIF-
1a was phosphorylated by MAPKERK1/2 and not by
MAPKp38 or MAPKJNK [172]. Interestingly, MAP-
KERK1/2 stoichiometrically phosphorylated HIF-1
MAPKERK1/2 in vitro, as judged by a complete upper
shift of HIF-1a. More importantly, it was demonstrat-
ed that the activation of the MAPKERK1/2 pathway in
quiescent cells induced the phosphorylation and shift
of HIF-1a, which was abrogated in the presence of the
MEK inhibitor, PD-98059.
In addition, with a VEGF promoter mutated at sites
shown to be MAPK-sensitive (SP-1/AP-2-88-66 site),
MAPKERK1/2 activation was sufficient to promote the
transcriptional activity of HIF-1 [172]. This interac-
tion between HIF-1a and MAPKERK1/2 indicated a
cooperation between hypoxic and growth factor sig-
nals that ultimately leads to the increase in HIF-1-
mediated gene expression. In support of the afore-
mentioned observations, it was also demonstrated that
in human microvascular endothelial cells-1 (HMEC-
1), ERK kinases were activated during hypoxia.
Using dominant negative mutants, Minet et al. [173,
174] concluded that MAPKERK1 was needed for HIF-
1 transactivation activity. Moreover, the same group
further showed that HIF-1a was phosphorylated in
hypoxia by a MAPKERK-dependent pathway, suggest-
ing a role for MAPK signaling in the transcriptional
response to hypoxia.
The elucidation of the molecular mechanisms gov-
erning the transition from a nonangiogenic to an
angiogenic phenotype is central for understanding
and controlling malignancies. Viral oncogenes repre-
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–1285 1267
sent powerful tools for disclosing transforming mech-
anisms, and they may also afford the possibility of
investigating the relationship between transforming
pathways and angiogenesis. In this regard, Sodhi et
al. [175,176] have recently observed that a constitu-
tively active G-protein-coupled receptor (GPCR)
encoded by the Kaposi’s sarcoma-associated herpes
virus (KSHV)/human herpes virus (HHV)-8 was
oncogenic and further stimulated angiogenesis by
increasing the secretion of VEGF, which is a key
angiogenic stimulator and a critical mitogen for the
development of Kaposi’s sarcoma. KSHV GPCR en-
hanced the expression of VEGF by stimulating the
activity of HIF-1a, which, in turn, activated transcrip-
tion from an HRE within the 5V-flanking region of the
VEGF promoter. Interestingly, stimulation of HIF-1a
by the KSHV GPCR involved the phosphorylation of
its regulatory/inhibitory domain by the MAPKp38 sig-
naling pathway, thereby enhancing its transcriptional
activity. Moreover, specific inhibitors of the MAPKp38
(SKF-86002) and MAPKERK1/2 (PD-98059) pathways
were able to inhibit the activation of the transactivating
action of HIF-1a induced by the KSHVGPCR, as well
as the VEGF expression and secretion in cells over-
expressing this receptor, suggesting that the KSHV
GPCR oncogene subverts convergent physiological
pathways leading to angiogenesis and provides insight
into a mechanism whereby growth factors and onco-
genes acting upstream from MAPK, as well as inflam-
matory cytokines and cellular stresses that activate
MAPKp38, can interact with the hypoxia-dependent
machinery of angiogenesis [175,176].
Oncogenic transformation and hypoxia induced
glut-1 mRNA (glucose transporter during hypoxia).
In this regard, the interaction between the Ras onco-
gene and hypoxia in upregulating glut-1 mRNA levels
using Rat1 fibroblasts transformed with H-Ras (Rat1–
Ras) was investigated. Transformation with H-Ras led
to a substantial increase in glut-1 mRNA levels under
normoxic conditions and additively increased glut-1
mRNA levels in concert with hypoxia [177]. Using a
luciferase reporter construct containing 6 kb of the
glut-1 promoter, it was shown that this effect was
mediated at the transcriptional level. Promoter activity
was much higher in Rat1–Ras cells than in Rat1 cells,
and could be downregulated by cotransfection with a
dominant negative Ras construct (Ras-N17). In addi-
tion, a 480-bp cobalt/hypoxia-responsive fragment of
the promoter containing a HIF-1 binding site showed
significantly higher activity in Rat1–Ras cells than in
Rat1 cells, suggesting that Ras might mediate its effect
through HIF-1 even under normoxic conditions.
Consistent with this, Rat1–Ras cells displayed
higher levels of HIF-1a protein under normoxic con-
ditions. In addition, a promoter construct containing a
4-bp mutation in the HIF-1 binding site showed lower
activity in Rat1–Ras cells than a construct with an
intact HIF-1 binding site. The activity of the latter
construct, but not the former, could be downregulated
by Ras-N17, supporting the significance of the HIF-1
binding site in regulation by Ras [177]. Of note, the
PI3K inhibitor LY29004 downregulated glut-1 pro-
moter activity and mRNA levels under normoxia and
also decreased HIF-1a protein levels. Furthermore, the
MAPKERK1/2 cascade, known to phosphorylate HIF-
1a, did not modulate the degradation/stabilization
profile of HIF-1a. However, recent evidence sug-
gested that the rate of HIF-1a degradation depends
on the duration of hypoxic stress. In this respect, the
degradation of HIF-1a was suppressed by: (i) inhibit-
ing general transcription with actinomycin D, or (ii)
specifically blocking HIF-1-dependent transcriptional
activity [178].
In keeping with these findings, it was postulated
that HIF-1a might be targeted to the proteasome via a
HIF-1a proteasome-targeting factor (HPTF), whose
expression was directly under the control of HIF-1-
mediated transcriptional activity. Although HPTF is
not yet molecularly identified, it is clearly distinct
from the pVHL. In preference to these observations,
recent results also indicated that the stabilization of
HIF-1a protein, by treatment of proteasome inhibi-
tors, was not sufficient for hypoxia-induced gene
activation, and an additional hypoxia-dependent mod-
ification was found necessary for gene expression by
HIF-1a [179]. In this respect, it was demonstrated that
PD-98059 did not change either the stabilization or
the DNA-binding ability of HIF-1a, but it inhibited
the transactivation ability of HIF-1a, thereby reducing
the hypoxia-induced transcription of both an endoge-
nous target gene and a hypoxia-responsive reporter
gene. Furthermore, hypoxia induced MAPKERK1/2
phosphorylation and the expression of dominant-neg-
ative MAPKERK1/2 mutants reduced HIF-1-dependent
transcription of the hypoxia-responsive reporter gene,
suggesting that the hypoxia-induced transactivation
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–12851268
ability of HIF-1a may be regulated by different
mechanisms than its stabilization and DNA-binding,
and that these processes can be, at least in part,
experimentally dissociated [179]. These observations
led to the conclusion that MAPKERK1/2 could regulate
the transactivation, but not the stabilization or DNA-
binding ability, of HIF-1a.
In support of this role of MAPK in HIF-1 stabi-
lization and DNA-binding activity, Tacchini et al.
[180] recently reported that hepatocyte growth factor
(HGF), a multifunctional cytokine of mesenchymal
origin, has the potential to activate the DNA binding
of HIF-1a in the HepG2 cell line. An increased
expression of HIF-1a (mRNA and nuclear protein
levels) was also observed. To investigate the molec-
ular basis of the HIF-1 response under this non-
hypoxic condition, the expression of putative target
genes was evaluated. It was found that a time-
dependent increase in steady-state mRNA levels of
heme oxygenase and urokinase plasminogen activator
followed by that of urokinase receptor was evident.
The enhanced expression of these genes might confer
the invasive phenotype, since HGF is a proliferative
and scatter factor [180]. In addition, HIF-1 activity
and its regulation in HGF-treated cells were fol-
lowed: (i) the activation of HIF-1 DNA binding
was prevented by proteasome blockade, probably
because stabilization of the cytosolic a-subunit pro-
tein level was not sufficient to generate a functional
form (also under these conditions, nuclear protein
level of HIF-1a did not increase); (ii) N-acetyl-L-
cysteine (NAC), a free radical scavenger and a GSH
precursor, strongly decreased HIF-1 activation, sug-
gesting a role of ROS in this process; and (iii) the
thiol-reducing agent dithiothreitol (DTT) was inef-
fective. Consistent with these observations, NAC
reduced the stimulatory effect of HGF on stress
kinase activities, while MAPKERK1/2 was unmodi-
fied, suggesting an involvement of MAPKJNK and
MAPKp38 in HIF-1 activation [180]. Of interest,
LY294002 caused the blockade of PI3K and pre-
vented the enhancement of HIF-1 DNA binding and
MAPKJNK activity; the inhibition of MAPKERK1/2
phosphorylation was rather ineffective.
Environmental signals in the cellular milieu such as
hypoxia, growth factors, extracellular matrix (ECM),
or cell surface molecules on adjacent cells can activate
signaling pathways that communicate the state of the
environment to the nucleus. Several groups have
evaluated gene expression or signaling pathways in
response to increasing cell density as an in vitro
surrogate for in vivo cell–cell interactions. These
studies have also perhaps assumed that cells grown
at various densities in standard in vitro incubator
conditions do not have different pericellular oxygen
levels [180,181]. However, pericellular hypoxia can be
induced by increasing cell density, which can exert
profound influences on the target cell lines and may
explain a number of findings previously attributed to
normoxic cell–cell interactions. Thus, the hypothesis
that cell–cell interactions, as evaluated by the surro-
gate approach of increasing in vitro cell density in
routine normoxic culture conditions, results in pericel-
lular hypoxia in prostate cancer cells was examined. A
recent report indicated that paracrine cell interactions
could induce nuclear localization of HIF-1a protein
and that this translocation was associated with strong
stimulation of the HRE reporter activity [181]. More-
over, cell density-induced activity of the HRE was
dependent on NO production, which acts as a diffus-
ible paracrine factor secreted by densely cultured cells,
suggesting that cell interactions associated with peri-
cellular hypoxia might lead to the physiological induc-
tion of HRE activity via the cooperative action of Ras,
MEK1, and HIF-1a via pericellular diffusion of NO.
The role of hypoxia and MAPK signaling pathways in
the regulation of HIF-1 is schematized in Fig. 9.
4.2. MAPK-mediated regulation of nuclear factor-jB(NF-jB)
Although the transcription factor NF-nB has been
originally recognized in regulating gene expression in
B-cell lymphocytes [182], subsequent investigations
have demonstrated that it is one member of a ubiqui-
tously expressed family of Rel-related transcription
factors that serve as critical regulators of many genes,
including those of proinflammatory cytokines [183–
201]. NF-nB comprises the Rel family of inducible
transcription factors that are key mediators in regulat-
ing the progression of the inflammatory process [202–
213]. Therefore, the activation and regulation of the
NF-nB/Rel transcription family, via nuclear transloca-
tion of cytoplasmic entities and complexes, play a
central role in the evolution of inflammation through
the regulation of genes essentially involved in encoding
Fig. 9. The role of hypoxia and MAPK signaling pathways in the regulation of HIF-1 and HIF-1-dependent gene transcription (see text for
further discussion).
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–1285 1269
proinflammatory cytokines and other inflammatory
mediators [214–227].
The NF-nB/Rel family includes five members: NF-
nB1 (p50/p105 {p50 precursor}), NF-nB2 (p52/p100
{p52 precursor}), RelA (p65), RelB (p68), and c-Rel
(p75). 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. The NF-nBmembers contain a Rel
homology domain (RHD), which is responsible for
dimer formation, nuclear translocation, sequence-spe-
cific consensus DNA recognition, and interaction with
inhibitory nB (InB) proteins, 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 recogni-
tion of the nuclear localization signal (NLS) by the
nuclear import machinery, thereby retaining the NF-
nB complex within the cytosol. In its inactive state,
the heterodimeric NF-nB, which is mainly composed
of two subunits, p50 (NF-nB1) and p65 (RelA), is
present in the cytoplasm associated with InB [225–
228]. Upon stimulation, such as with cytokines and
lipopolysaccharide endotoxin (LPS), derived from the
cell wall of Gram-negative bacteria, InB-a, the major
cytosolic inhibitor of NF-nB, undergoes phosphory-
lation on serine/threonine residues, ubiquitination, and
Fig. 10. NF-nB signal transduction pathway. Various incoming
signals converge on the activation of the InB kinase (IKK) complex.
IKK then phosphorylates InB at two N-terminal serines, which
signals it for ubiquitination and proteolysis (proteasome). Freed NF-
nB complex (p50/p65) enters the nucleus and activates gene
expression.
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–12851270
subsequent proteolytic degradation, thereby unmask-
ing the NLS on p65 and allowing nuclear transloca-
tion of the complex. This sequential propagation of
signaling ultimately results in the release of NF-nBsubunits from the InB-a inhibitor, allowing translo-
cation 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 ki-
nase (NIK) and MEKK-1, both of which are involved
in the activation of InB kinases, IKK1 and IKK2,
components of the IKK signalsome. These kinases
phosphorylate members of the InB family, including
InB-a, the major cytosolic inhibitor of NF-nB, at
specific serines within their amino termini, thereby
leading to site-specific ubiquitination and degradation
by the proteasome. This sequential trajectory culmi-
nating in the inducible degradation of InB, which
occurs through consecutive steps of phosphorylation
and ubiquitination, allows freeing of the NF-nBcomplex, which translocates onto the nucleus, binds
specific nB moieties, and initiates gene transcription
(Fig. 10).
Hypoxia can cause the activation of NF-nB and the
phosphorylation of its inhibitory subunit, InB-a. Forinstance, Beraud et al. [228] reported that hypoxia,
reoxygenation, and the tyrosine phosphatase inhibitor
pervanadate activate NF-nB via a mechanism involv-
ing the phosphorylation of InB-a on tyrosine residues.
This modification, however, did not lead to degrada-
tion of InB-a by the proteasome/ubiquitin pathway, as
evident from the stimulation of cells with proinflam-
matory cytokines. It was shown that p85-a, the regu-
latory subunit of phosphatidylinositol 3-kinase,
specifically associates through its Src homology 2
domains with tyrosine-phosphorylated InB-a in vitro
and in vivo after stimulation of T cells with pervana-
date [227]. This association could provide a mecha-
nism by which newly tyrosine-phosphorylated InB-ais sequestered from NF-nB.
Another mechanism by which phosphatidylinositol
3-kinase contributed to NF-nB activation in response
to pervanadate appeared to involve its catalytic p110
subunit. This was evident from the inhibition of
pervanadate-induced NF-nB activation and reporter
gene induction by treatment of cells with nanomolar
amounts of a phosphatidylinositol 3-kinase inhibitor,
Wortmannin. The compound had virtually no effect on
TNF- and IL-1-induced NF-nB activities. In addition,
Wortmannin did not inhibit the tyrosine phosphoryla-
tion of InB-a or alter the stability of the phosphatidy-
linositol 3-kinase complex, but inhibited Akt kinase
activation in response to pervanadate, suggesting that
both the regulatory and the catalytic subunit of phos-
phatidylinositol 3-kinase play a role in NF-nB acti-
vation by the tyrosine phosphorylation-dependent
pathway [228].
These observations were also supported by Canty
et al. [229] who reported assays that also showed
substantial NF-nB activation in hypoxic HUVECs
after reoxygenation and in cultures treated with
H2O2. Pervanadate also induced marked NF-nB acti-
vation in HUVECs, indicating that H2O2-induced NF-
nB activation is potentiated by the inhibition of
tyrosine phosphatases. Western blotting of cytoplas-
mic InB-a demonstrated that NF-nB activation in-
duced by oxidative stress was not associated with InB-a degradation. In contrast, however, TNF-a-induced
NF-nB activation occurred in concert with InB-a
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–1285 1271
degradation. Furthermore, inhibition of InB-a degra-
dation with a proteasome inhibitor, MG-115, blocked
NF-nB activation induced by TNF-a; however, MG-
115 had no effect on NF-nB activation during oxida-
tive stress.
With the use of dominant negative mutants of
Ha-Ras and Raf-1, Koong et al. [230] investigated
some of the early signaling events leading to the activa-
tion of NF-nB by hypoxia. Both dominant negative
alleles of Ha-Ras and Raf-1 inhibited NF-nB induction
by hypoxia, suggesting that the hypoxia-induced
pathway of NF-nB induction is dependent on Ras
and Raf-1 kinase activity. Furthermore, although con-
ditions of low oxygen can also activate MAPKERK1/2,
these kinases did not appear to be involved in regu-
lating NF-nB by hypoxia, an observation supported by
the notion that the dominant negative mutants of
MAPK had no inhibitory effect on NF-nB activation
by hypoxia [230]. Moreover, an increase in Src proto-
oncogene activity of cellular exposure to hypoxia was
observed under similar conditions. It was subsequently
postulated that Src activation by hypoxia might be one
of the earliest events that precedes Ras activation in the
signaling cascade that ultimately leads to the phos-
phorylation and dissociation of the inhibitory subunit
of NF-nB, InB-a.Similarly, the inhibitor of MAPKERK1/2 (PD-98059)
blocked CoCl2-induced NF-nB activation and VCAM-
Fig. 11. The role of hypoxia and MAPK signaling pathways in the r
1 expression (CoCl2 acts as a mimetic molecule for
hypoxia to study cellular signaling pathways) [231].
These aforementioned mechanisms were reinforced
with observations related to a stress-induced cytokine,
termed cardiotrophin-1 (CT-1), which belongs to the
IL-6/glycoprotein 130 receptor-coupled cytokine fam-
ily. CT-1 is released from the heart in response to
hypoxic stress and it protects cardiac myocytes from
hypoxia-induced apoptosis, thus establishing a central
role for this cytokine in the cardiac stress response
[232]. It was observed that, in cardiac myocytes, CT-1
activated MAPKp38 and MAPKERK as well as Akt. CT-
1 also induced the degradation of the NF-nB cytosolic
anchor, InB, as well as the translocation of the RelA
(p65) subunit (the major transactivating member of the
Rel family) of NF-nB to the nucleus and increased
expression of an NF-nB-dependent reporter gene.
Furthermore, inhibitors of the MAPKp38, MAPKERK,
or Akt pathways each partially reduced CT-1-mediated
NF-nB activation, as well as the cytoprotective effects
of CT-1 against hypoxic stress. Together, the inhibitors
completely blocked CT-1-dependent NF-nB activation
and cytoprotection, indicating that CT-1 signals
through MAPKs and Akt in a parallel manner to
activate NF-nB. The role of hypoxia and MAPK
signaling pathways in the regulation of NF-nB and
NF-nB-dependent gene transcription is schematized in
Fig. 11.
egulation of NF-nB and NF-nB-dependent gene transcription.
Fig. 12. The role of hypoxia and MAPK signaling pathways in the
regulation of AP-1 and AP-1-dependent gene transcription via the
cytokine-dependent pathway.
nopharmacology 4 (2004) 1249–1285
4.3. MAPK-mediated regulation of activating
protein-1 (AP-1)
Pathophysiological hypoxia is an important mod-
ulator of gene expression in solid tumors and other
pathologic conditions. In this respect, it was ob-
served that transcriptional activation of the c-jun
proto-oncogene in hypoxic tumor cells correlates
with the phosphorylation of the ATF-2 transcription
factor [233]. This finding suggested that hypoxic
signals transmitted to c-jun involve protein kinases
that target AP-1 complexes (c-Jun and ATF-2) that
bind to its promoter region. Stress-inducible protein
kinases capable of activating c-jun expression in-
clude MAPKJNK and MAPKp38. Transient activation
of SAPK/JNKs occurred by tumor-like hypoxia
concurrent with the transcriptional activation of
MKP-1, a stress-inducible member of the MAPK
phosphatase (MKP) family of dual-specificity pro-
tein tyrosine phosphatases. Of interest is the obser-
vation that Northern blots showed an increase in the
level of c-jun and c-fos subunits during hypoxia.
Gel mobility shift analysis of nuclear extracts from
hypoxia-exposed cells showed an increase in AP-1
binding activity [234]. In addition, hypoxic treat-
ment strongly activated MAPKJNK-1, thereby lead-
ing to phosphorylation and activation of c-Jun.
Furthermore, expression of a dominant negative
mutant of MAPKJNK-1 suppressed hypoxia-induced
MAPKJNK-1 activation as well as reporter gene
expression.
Unequivocally, using antisense c-fos strategy, it
was shown that c-fos is essential for the activation of
AP-1 complex and subsequent stimulation of down-
stream genes [235]. Furthermore, hypoxia caused
Ca2 + influx through L-type voltage-gated Ca2 +
channels and the hypoxia-induced c-fos gene expres-
sion was Ca2 +/calmodulin-dependent. It was also
demonstrated that hypoxia induced the activation of
MAPKERK and MAPKp38, but not MAPKJNK, con-
trary to observations of Le and Corry [234]. The
phosphorylation of MAPKERK was found to be
essential for c-fos activation via serum response
element (SRE) cis-element. Further characterization
of nuclear signaling pathways provided evidence for
the involvement of Src, a nonreceptor PTK, and Ras,
a small G-protein, in the hypoxia-induced c-fos gene
expression, suggesting a possible role for nonrecep-
J.J. Haddad / International Immu1272
tor PTKs in propagating signals from G-protein-
coupled receptors to the activation of immediate-
early genes such as c-fos during hypoxia.
In cancerous HeLa cells, hypoxic conditions
have been reported to induce the transcriptional
activation of c-fos transcription via the SRE [236].
Mutations in the binding site for the ternary com-
plex factor Elk-1 and SRE abolished this induction,
indicating that a ternary complex at the SRE is
necessary for the induction of the c-fos gene under
hypoxia. The transcription factor Elk-1 was cova-
lently modified by phosphorylation in response to
hypoxia. Furthermore, this hyperphosphorylation of
Elk-1, the activation of MAPK, and the induction
of c-fos transcripts were blocked by PD-98059. An
in vitro kinase assay with Elk-1 as substrate
showed that MAPK is activated under hypoxia.
Furthermore, the activation of MAPK corresponded
temporally with the phosphorylation and activation
of Elk-1 [236]. On mechanisms, a decrease of
intracellular ROS level by hypoxia induced c-fos
via the MAPK pathway, suggesting that the intra-
cellular redox levels may be directly coupled to
tumor growth, invasion, and metastasis via Elk-1-
dependent induction of c-fos-controlled genes
[236,237]. The role of hypoxia and MAPK signal-
ing pathways in the regulation of AP-1 is shown in
Fig. 12.
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–1285 1273
5. The role of MAPK signaling pathways in
hypoxia- or anoxia-tolerant organisms
Freshwater turtles, such as the western-painted
turtles Chrysemys picta bellii and the hatchling red-
eared turtles Trachemys scripta elegans, are consid-
ered among the most anoxia-tolerant, air-breathing
vertebrates [238–243]. Integrative and sustained
Fig. 13. Hypoxia-mediated regulation of MAPKp38 signaling pathway. (A) E
did not significantly affect the phosphorylation/activation of MAPKp38, as
h variably affected the phosphorylation/activation of MAPKp38, with suppre
MAPKp38 suppression was not observed at either 3 days or 6 weeks of hypo
form of MAPKp38, as verification for semiquantitative loading in parall
phosphorylated form of MAPKp38 under hypoxia/reoxygenation (6 h). (Th
other values were expressed relative to this unit.) ND, not determined;
experiments performed for each variable.
adaptations on the imposition of submergence anoxia
underlie the animal’s capacity to tolerate these con-
ditions for long periods of time [244–246]. C. picta
bellii and T. scripta elegans are unusually tolerant of
anoxia in that they survive 24–48 h of anoxia at 25
jC and 4–5 months at 2–3 jC during winter
dormancy. Survival of neurons in these remarkable
turtles involves a profound reduction in energy me-
xposure to hypoxia for a period of time ranging from 5 h to 6 weeks
compared with normoxia control. (B) Hypoxia/reoxygenation for 6
ssion at 1 day and 1 week of hypoxia followed by oxygenation. This
xia. The lower panel shows the expression of the nonphosphorylated
el lanes. (C) Histogram analysis of the relative abundance of the
e level of expression at normoxia was adjusted to one unit and all
n= 2–5, which designates the number of turtles and independent
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–12851274
tabolism to approximately 10–20% of the normoxic
rate at the same temperature [245–247], suggesting a
coordinated reduction of ATP-generating mechanisms
and ATP-consuming pathways. This metabolic ‘arrest’
has been shown to lead to suppression of ion channels,
thereby allowing decreased excitability, reduced ion
Fig. 14. Hypoxia-mediated regulation of MAPKp44/p42 (MAPKERK1/2) sign
from 5 h to 6 weeks variably and in a biphasic manner affected the phos
control. Whereas 5 h of hypoxia significantly increased the phosphorylatio
different from normoxia, and at 6 weeks of hypoxia, the phosphorylat
normoxia and 5 h. (B) Hypoxia/reoxygenation for 6 h mildly affected the
weeks of hypoxia followed by oxygenation. This MAPKp44/p42 suppressio
shows the expression of the nonphosphorylated form of MAPKp44/p42,
Histogram analysis of the relative abundance of the phosphorylated form
expression at normoxia was adjusted to one unit and all other values were
designates the number of turtles and independent experiments performed
translocation, and preservation of [ATP] during the
energetic stress imposed by anaerobic conditions
[248]. Furthermore, other targets have been sup-
pressed, including numerous enzymes and molecules
that regulate protein synthesis [249,250]. Another
feature that characterizes survival is the ability to
aling pathway. (A) Exposure to hypoxia for a period of time ranging
phorylation/activation of MAPKp44/p42, as compared with normoxia
n/activation of MAPKp44/p42, 1 day to 1 week was not significantly
ion/activation of MAPKp44/p42 was suppressed, as compared with
phosphorylation/activation of MAPKp44/p42, with suppression at 6
n was not observed at 1 day to 1 week of hypoxia. The lower panel
as verification for semiquantitative loading in parallel lanes. (C)
of MAPKp44/p42 under hypoxia/reoxygenation (6 h). (The level of
expressed relative to this unit.) ND, not determined; n= 2–5, which
for each variable.
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–1285 1275
buffer an acid–base equilibrium in response to lactate
accumulation due to anaerobic glycolysis [250]. This
latter mechanism is centered on the release of carbo-
nates from the bone and shell to enhance body fluid
buffering of lactic acid. Therefore, the combination of
slow metabolic activity and responsive mineral re-
serve is crucial to the survival of those animals under
unprecedented conditions.
The biochemical and physiological mechanisms of
anoxia tolerance in turtles have been previously ex-
amined at the level of ion transport and ATP turnover
to better understand the effect of oxygen deprivation
[251]. However, changes in the phosphorylation state
of key enzymes and kinases may occur during anoxia;
therefore, reversible protein phosphorylation could be
a critical factor and major mechanism of metabolic
reorganization for enduring anaerobiosis [251,252].
For instance, it has been shown that anoxia mediated
changes in the activities of PKA, PKC, and protein
Ppase-1 [253]. Furthermore, anoxia was shown to
impose variations in protein synthesis, mRNA accu-
Fig. 15. Hypoxia-mediated regulation of antiapoptotic cofactor, Bcl-2, and
time ranging from 5 h to 6 weeks mildly upregulated the protein expression
control. (B) Hypoxia/reoxygenation for 6 h has no effect on Bcl-2 expressio
weeks upregulated the protein expression of Bax at 3 days and 1 wee
reoxygenation for 6 h variably affected Bax expression, with upregulation
mulation, and gene transcription in turtle organs,
suggesting that upregulation of selective genes is
crucial for surviving anoxia.
The role of two vertebrate MAPKs in mediating
responses to in vivo anoxia or freezing exposures was
examined in four organs (liver, heart, kidney, and
brain) of hatchling red-eared turtles, T. scripta elegans,
which are naturally tolerant of these stresses [250,251].
The extracellular signal-regulated kinases (MAP-
KERK1/2) were not stress-activated except in the brain
of frozen turtles. MAPKJNK was transiently activated
by anoxia exposure in all four organs (after 1 h in brain
or 5 h in other organs), but activity was suppressed
during freezing except in the brain, which showed a
transient activation of MAPKJNK after 1 h. In addition,
changes in the concentrations of the transcription
factors, c-fos and c-myc, were also stress- and organ-
specific. These results for an anoxia-tolerant animal
suggested the potential importance of the MAPKs and
immediate-early genes (c-fos, c-myc) in mediating
adaptive responses to oxygen deprivation.
proapoptotic cofactor, Bax. (A) Exposure to hypoxia for a period of
of Bcl-2 at 1 day to 1 week of hypoxia, as compared with normoxia
n. (C) Exposure to hypoxia for a period of time ranging from 5 h to 6
k of hypoxia, as compared with normoxia control. (D) Hypoxia/
at 1 day, 3 days, and 1 week of hypoxia followed by oxygenation.
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–12851276
Recently, Haddad et al. have investigated in vivo
the hypoxia-mediated regulation of MAPK signaling
pathways and caspase/apoptosis cofactor expression
in anoxia-tolerant turtles, C. picta bellii (unpublished
observations). Whereas hypoxia has no apparent ef-
fect on MAPKp38 phosphorylation, hypoxia–reoxy-
genation suppressed this pathway in early hypoxia
(Fig. 13). In contrast, hypoxia-mediated phosphoryla-
tion of MAPKERK followed a biphasic module in that
there was enhancement at early hypoxia followed by
suppression at late hypoxia, similar to the effect
observed with MAPKJNK (Fig. 14). Of interest, neither
hypoxia nor hypoxia–reoxygenation mediated the
activation of the caspase pathway; however, hypoxia
upregulated the expression of Bax and, to a lesser
extent, Bcl-2, an effect potentiated with reoxygenation,
indicating the involvement of a Bax-sensitive pathway
(Fig. 15) (Haddad et al., unpublished observations,
UCSF). In summary, these results indicate that the
regulation of MAPK signaling pathways in anoxia-
tolerant turtles is inharmonious, and that apoptosis
regulation is caspase-insensitive and requires, at least
in part, the involvement of a Bax-dependent mecha-
nism. The patterns of MAPK activation in a stress-
tolerant animal suggest the relative importance of these
kinase pathways in cellular adaptation to oxygen
deprivation or freezing and identify novel natural
activators of MAPKs in vivo. The specificity of the
signaling pathways is also emphasized here as general
whole body stresses (anoxia and freezing) activated
Fig. 16. Hypoxia-mediated regulation of MAPK signaling pathways
in anoxia-tolerant cell models, such as the western-painted turtle C.
picta bellii and the hatchling red-eared turtle T. scripta elegans.
individual MAPKs in a tissue-, time-, and stress-
dependent manner. Hypoxia-mediated regulation of
MAPK signaling in anoxia-tolerant turtles is shown
in Fig. 16.
6. Summary, conclusion, and future prospects
Hypoxia is a crucial signal in development and
physiology, but it may well be a key element in the
development of many abnormalities and disease
conditions [254,255]. The complex signaling path-
ways mediated by hypoxia revolve around the hinge
of controlling gene transcriptions by upstream effec-
tors and regulators. This feedforward mode of gene
regulation involves the revolving axis of MAPK
signaling molecules, which coordinate messages
from cell receptors to effector cofactors situated at
different levels within the intriguing hierarchy of cell
regulation. Not only are MAPKs regulated by hyp-
oxia signaling, but also they play a crucial role in
mediating hypoxia-dependent gene regulation, hence
the evolution of a revolving axis for cell regulation
[256–259]. In so far as the underlying mechanisms
controlling this multifaceted revolving axis of gene
regulation are vaguely comprehended, the basis for
complex interactions between MAPK modules and
the various components of the cell machinery has
already paved the way to developing a strategy for
understanding not only the processes of development
but also the evolution of pathophysiologic conditions
stemming out of the malfunctioning of these sys-
tems. The burgeoning, yet central, role of hypoxia
MAPK modules as a major determinant of many
life-critical cell functions may perhaps allow us to
understand the complex mechanisms in display me-
diating the regulation of transcription and gene
expression for better therapeutic pharmacologic inter-
ventions [260–272].
Acknowledgements
The author’s work is financially supported by the
Anonymous Trust (Scotland), the National Institute
for Biological Standards and Control (England), the
Tenovus Trust (Scotland), the UK Medical Research
Council (MRC, London), the Wellcome Trust (Lon-
J.J. Haddad / International Immunopharmacology 4 (2004) 1249–1285 1277
don), and the National Institutes of Health (NIH;
USA). Dr. John Haddad held the Georges John
Livanos (London) and the NIH (UCSF, California,
USA) award fellowships. This manuscript was written
at UCSF when the author was a research fellow. The
author appreciatively thanks Professor John Hayes
(Biomedical Research Center, University of Dundee,
Scotland, UK) for critical reading of the manuscript,
and Mrs. Jennifer Schuyler (Department of Anesthesia
and Perioperative Care, UCSF) for her expert editorial
assistance.
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