review article hypoxia and the regulation of mitogen...

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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: [email protected] (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.


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].


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


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


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.


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-


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.


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.


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-


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-


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).


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.


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


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.


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-


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-


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


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).


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.


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


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.


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


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.


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.


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-


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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